Method for producing carotenoids or their precursors using genetically modified organisms of the blakeslea genus, carotenoids or their precursors produced by said method and use thereof

ABSTRACT

The invention relates to a method for producing carotenoids or their precursors using genetically modified organisms of the  Blakeslea  genus. Said method comprises the following steps (i) transformation of at least one of the cells, (ii) optional homokaryotic conversion of the cells obtained in step (i) to produce cells, in which one or more genetic characteristics of the nuclei are all modified in an identical manner and said modification manifests itself in the cells, (iii) selection and reproduction of the genetically modified cell or cells, (iv) cultivation of the genetically modified cells, (v) preparation of the carotenoids produced by the genetically modified cells or the carotenoid precursor produced by said genetically modified cells. The invention also relates to carotenoids or their precursors produced according to said method and to the use thereof.

The invention relates to a method for producing carotenoids or theirprecursors using genetically modified organisms of the Blakeslea genus,to carotenoids or their precursors produced by said method and to theuse and provision thereof, in particular as highly pure carctenoids, asfoodstuffs comprising carotenoid-producing organisms and at least onecarotenoid, in particular animal feedstuffs, animal feed supplements andfood supplements, and to the use of the carotenoids obtainable by saidmethod for producing cosmetic, pharmaceutical, dermatologicalpreparations, foodstuffs or food supplements.

Blakeslea trispora is a known producer organism for β-carotene (Ciegler,1965, Adv Appl Microbiol. 7:1) and lycopene (EP 1201762, EP 1184464, WO03/038064).

Various DNA sequences of Blakeslea trispora are known already, inparticular the DNA sequence coding for the genes of carotenoidbiosynthesis from geranylgeranyl pyrophosphate to β-carotene (WO03/027293).

The high productivity achieved by Blakeslea for producing of lycopeneand β-carotene, in particular, renders this organism suitable forfermentative production of carotenoids.

It is also of interest to further increase the productivities ofcarotenes and their precursors which have previously been producednaturally and to enable further carotenoids such as, for example,xanthophylls to be produced which have been produced by and isolatedfrom Blakeslea only to a very low extent, if at all, previously.

Carotenoids are added to feedstuffs, foodstuffs, food supplements,cosmetics and medicaments. Carotenoids are used especially as pigmentsfor coloring. Aside from this, the antioxidative action of carotenoidsand other properties of these substances are utilized. The carotenoidsare divided into the pure hydrocarbons, the carotenes and theoxygen-containing hydrocarbons, the xanthophylls. Xanthophylls such ascanthaxanthin and astaxanthin are employed, for example, in thepigmentation of hens' eggs and fish (Britton et al. 1998, Carotinoids,Vol. 3, Biosynthesis and Metabolism). The carotenes β-carotene andlycopene are employed especially in human nutrition. β-Carotene, forexample, is used as a colorant for beverages. Lycopene hasdisease-preventing action (Argwal and Rao, 2000, CMAJ 163:739-744; Raoand Argwal 1999, Nutrition Research 19:305-323). The colorlesscarotenoid precursor phytoene is especially suitable for applications asantioxidant in cosmetic, pharmaceutical or dermatological preparations.

Most of the carotenoids and their precursors which are employed asadditives in the abovementioned applications are prepared by chemicalsynthesis. Said chemical synthesis is very complicated and causes highproduction costs. In contrast, fermentative processes are comparativelysimple and based on inexpensive starting materials. Fermentativeprocesses to produce carotenoids and their precursors may beeconomically attractive and capable of competing with chemicalsynthesis, if the productivity of previous fermentative processes wereincreased or new carotenoids were able to be prepared on the basis ofthe known producer organisms.

This requires a genetically engineered, i.e. specific genetic,modification of Blakeslea, in particular if xanthophylls are to beproduced, since these compounds are not naturally synthesized by theBlakeslea wild type.

For example, two previous methods are known for producing phytoene bymeans of fermentation of Blakeslea trispora:

-   -   (i) Random mutagenesis using chemical agents such as MNNG may        generate mutants which cannot convert phytoene to lycopene, and        thus further to β-carotene (Mehta and Cerdá-Olmedo, 1995, Appl.        Microbiol. Biotechnol. 42:836-838).    -   (ii) Addition of inhibitors of the enzyme phytoene desaturase,        such as, for example, diphenylamine and cinnamyl alcohol, can        block further conversion of phytoene, causing the latter to        accumulate (Cerdá-Olmedo, 1989, In: E. Vandamme, ed.        Biotechnology of vitamin, growth factor and pigment production.        London: Elsevier Applied Science, pp. 27-42).

The methods mentioned for preparing phytoene using Blakeslea trispora,however, have a number of disadvantages.

Said random mutagenesis usually affects not only the genes of carotenoidbiosynthesis for further conversion of phytoene but also other importantgenes. For this reason, growth and synthetic performance of the mutantsare often impaired. The generation of, for example, phytoeneoverproducers by random mutagenesis of lycopene overproducers orβ-carotene overproducers can therefore be achieved only with greatexperimental complexity, if at all. The addition of inhibitors increasesproduction costs and may cause a contamination of the product. Inaddition, cell growth may be impaired by the inhibitor, thus limitingproduction of carotenoids or their precursors, in particular phytoene.

The abovementioned disadvantages of random mutagenesis and addition ofinhibitor could be avoided by a genetically engineered modification.

Thus far, however, no methods for the genetically engineered, i.e.specific genetic, modification of Blakeslea, in particular Blakesleatrispora, are known.

A method for the production of genetically modified fungi which has beensuccessfully employed in some cases is Agrobacterium-mediatedtransformation. Thus, for example, the following organisms have beentransformed by agrobacteria: Saccharomyces cerevisiae (Bundock et al.,1995, EMBO Journal, 14:3206-3214), Aspergillus awamori, Aspergillusnidulans, Aspergillus niger, Colletotrichum gloeosporioides, Fusariumsolani pisi, Neurospora crassa, Trichoderma reesei, Pleurotus ostreatus,Fusarium graminearum (van der Toorren et al., 1997, EP 870835),Agraricus bisporus, Fusarium venenatum (de Groot et al., 1998, NatureBiotechnol. 16:839-842), Mycosphaerella graminicola (Zwiers et al. 2001,Curr. Genet. 39:388-393), Glarea lozoyensis (Zhang et al., 2003, Mol.Gen. Genomics 268:645-655), Mucor miehei (Monfort et al. 2003, FEMSMicrobiology Lett. 244:101-106).

Of particular interest is a homologous recombination which involves asmany sequence homologies as possible between the DNA to be introducedand the cellular DNA, so that it is possible to introduce or eliminatesite-specifically genetic information in the genome of the recipientorganism. Otherwise, the donor DNA will be integrated into the genome ofthe recipient organism by illegitimate or nonhomologous recombinationwhich is not site-specific.

Agrobacterium-mediated transformation and subsequent homologousrecombination of the transferred DNA have been detected previously forthe following organisms: Aspergillus awamori (Gouka et al. 1999, NatureBiotech 17:598-601), Glarea lozoyensis (Zhang et al., 2003, Mol. Gen.Genomics 268:645-655), Mycosphaerella graminicola ((Zwiers et al. 2001,Curr. Genet. 39:388-393).

Another known method for transforming fungi is electroporation. Hill,Nucl. Acids. Res. 17:8011 has shown the integrative transformation ofyeast by electroporation. Transformation of filamentous fungi has beendescribed by Chakaborty and Kapoor (1990, Nucl. Acids. Res. 18:6737).

A “biolistic” method, i.e. the transfer of DNA by bombardment of cellswith DNA-loaded particles, has been described, for example, forTrichoderma harzianum and Gliocladium virens (Lorito et al. 1993, Curr.Genet. 24:349-356).

However, it has not been possible previously to successfully employthese methods for specific genetic modification of Blakeslea and inparticular Blakeslea trispora.

A particular difficulty in producing genetically modified Blakeslea andBlakeslea trispora is the fact that their cells are multinuclear at allstages of the sexual and vegetative cell cycles. For example, spores ofthe Blakeslea trispora strains NRRL2456 and NRRL2457 were found to havean average of 4.5 nuclei per spore (Metha and Cerdá-Olmedo, 1995, Appl.Microbiol. Biotechnol. 42:836-838). As a consequence of this, thegenetic modification is usually present only in one or a few nuclei,i.e. the cells are heterokaryotic.

If the genetically modified Blakeslea, in particular Blakeslea trispora,are intended to be used for production, it is important, in particularin the case of gene deletion, that the genetic modification is presentin all nuclei of the producer strains so as to make possible a stableand high synthetic performance without byproducts. The strains mustconsequently be homokaryotic with respect to said genetic modification.

A method of generating homokaryotic cells has been described only forPhycomyces blakesleeanus (Roncero et al., 1984, Mutat. Res. 125:195).According to the method described there, nuclei are eliminated in thecells by adding the mutagenic agent MNNG(N-methyl-N′-nitro-N-nitrosoguanidine) so as to obtain statistically acertain number of cells with only one functional nucleus. The cells arethen subjected to a selection in which only mononuclear cells having arecessive selection marker can grow into a mycelium. The progeny ofthese selected cells are multinuclear and homokaryotic. An example of arecessive selection marker for Phycomyces blakesleanus is dar. dar⁺strains absorb the toxic riboflavin analog 5-carbon-5-deazariboflavin,unlike dar⁻ strains (Delbrück et al. 1979, Genetics 92:27). Recessivemutants are selected by adding 5-carbon-5-deazariboflavin (DARF).

However, this method is unknown for Blakeslea, in particular Blakesleatrispora, and has in particular not been described in relation to atransformation or production of carotenoids or their precursors.

Isolation from natural resources is also carried out. A known example ofobtaining phytoene is to extract a mixture of carotenoids, vitamin E andother components, which also contains phytoene, from tomatoes, carrotsor palm oil etc. A problem here is the separation of the individualcarotenoids from one another. Thus, for example, phytoene cannot beobtained in a pure form by this method. In particular, the naturallyoccurring amount of carotenoids in the plants is low.

In contrast, fermentative processes are comparatively simple and basedon inexpensive starting materials. Fermentative processes to producecarotenoids may be economically attractive and capable of competing withchemical synthesis, if the productivity of previous fermentativeprocesses were increased or new carotenoids were able to be prepared onthe basis of the known producer organisms. A problem of the fermentativeproduction of carotenoids, however, are the work-up processes whichprovide only small amounts of highly pure carotenoids. Moreover, theyusually require processes with multiple steps, if appropriate with theuse of large amounts of solvents. Thus, large amounts of waste areproduced or a lot of effort has to go into the recycling process.

The production of carotenoids by various microorganisms is known per se.Thus, for example, WO 00/13654 A2 discloses the extraction of a mixtureof phytoene and phytofluene from algae of the species Dunaliella sp.This method too, does not produce phytoene in a pure form, and thelatter must be separated from the other products. Moreover, the algaeare genetically unmodified and their biosynthesis must be influenced bymeans of an added inhibitor.

WO 98/03480 A1 also discloses Blakeslea trispora as producer organismfor β-carotene. Here, β-carotene crystals are obtained from Blakesleatrispora biomass by means of extraction. However, the method describedrequires large amounts of different solvents in order to obtain crystalswith high purity by several extraction and washing steps. The amounts ofβ-carotene obtained are also small based on the amount of biomass used.

WO 01/83437 A1 discloses a method for extracting astaxanthin from yeast,which comprises treating the culture broth with microwave radiation forsterilization and cell disruption. According to this, cell disruption bymeans of microwave radiation is required in order to obtain astaxanthinfrom yeast without destroying it. Subsequently, astaxanthin is to beextracted by means of methanol, ethanol or acetone or mixtures thereof.This, however, requires large amounts of solvent (5 to 20 parts ofsolvent to 1 part of suspension) and a long time (24 h). Moreover,astaxanthin purities are not indicated and the amounts obtained aresmall. However, experiments of the applicant and other publicationsconfirm that extraction by means of methanol or ethanol is not possible.

WO 98/50574 likewise discloses the isolation of carotenoid crystals froma microorganism biomass, and here, in contrast to WO 01/83437 A1, it ispossible to use methanol, ethanol, acetone only for removing lipids fromthe biomass, i.e. for washing. Accordingly, the solvent used forextracting carotenoids is ethyl acetate, hexane or an oil. Subsequently,a plurality of purification and washing steps with large amounts ofethanol and water are required, resulting in a purity of only 93.3% witha yield of 35%.

WO 03/038064 A2 describes the fermentative production of lycopene bycocultivation of mutated Blakeslea trispora mating type (−) andBlakeslea trispora mating type (+) which produce lycopene withoutaddition of inhibitors of carotenoid biosynthesis. The mutant employedfor fermentation is generated by nonselective chemical mutation andsubsequent screening. The culture broth is worked up by means of celldisruption and subsequent purification with different aqueous media withvarying salt content and pH and with water-immiscible organic solventssuch as ethyl acetate, hexane and 1-butanol, in order to remove lipids.An extraction using large amounts of ethyl acetate is described as analternative. Information about the purity is missing. Since ethylacetate and hexane are solvents for lycopene, it can be assumed thatpart of the lycopene is washed out, thus reducing the theoreticallypossible yield.

WO 01/55100 A1 also describes the isolation of carotenoids in general,and specifically β-carotene, from the biomass by applying a plurality ofwashing and purification steps to the disrupted biomass withoutextraction by means of solvents. This involves washing disruptedBlakeslea trispora biomass with water, lye, acid, butanol and ethanol sothat the use of a large number of different solvents and aqueous mediais required. The purity of the β-carotene obtained is 96-98%. However,there is no information regarding the yield.

WO 97/36996 A2 generally describes a method for isolating substances(inter alia carotenoids) from microorganisms, said substances beingisolated from the biomass by means of solid/liquid extraction. Celldisruption is apparently not required here but the biomass must first berendered granulated and porous by extrusion. The possibility ofisolating only carotenoids and information about their purity or yieldare not indicated. The residue from the extrusion may subsequently beused as feed additive.

In all the methods described above, large amounts of solvent must beused for extraction, in order to increase the amount of isolatedcarotenoid by complete extraction, and/or large amounts of aqueous mediamust be used for purification and washing. This causes high costs andcomplicated recycling measures and, if appropriate, waste.

Moreover, the nutritious culture broth and the biomass present thereinare treated as waste, after extraction or isolation of the carotenoids.Aside from these superficial disadvantages, the methods indicated abovehave another decisive disadvantage, namely the fact that the carotenoidsmust be added to the foodstuffs subsequently, i.e. they are not part ofthe foodstuffs per se or are present only in an insufficient amount. Itwould therefore be greatly advantageous if the carotenoid content in thefoodstuffs would already be covered by the actual foodstuffs themselves.

Likewise, it is necessary to further increase the productivities of thepreviously naturally produced carotenes and their precursors and toenable further carotenoids such as, for example, xanthophylls,particularly preferably astaxanthin or zeaxanthin, and phytoene or bixinto be produced which have previously been produced by and isolated fromthe wild types of the microorganisms only to a very low extent, if atall.

It is an object of the invention to provide genetically modified cellsof Blakeslea strains, in particular Blakeslea trispora, which producecarotenoids or their precursors, in particular xanthophylls,particularly preferably astaxanthin or zeaxanthin, and phytoene orbixin. Moreover, the method is intended to allow carotenoid productivityof the modified cells to be increased compared to the corresponding wildtypes. The method is also intended to allow new cells or myceliumcomposed thereof to be generated which are suitable for the use in theproduction of carotenoids or their precursors which were previouslyunobtainable from the naturally occurring fungi in economicallyinteresting quantities, in particular xanthophylls, particularlypreferably astaxanthin or zeaxanthin, and phytoene or bixin. In thiscontext, the method is intended to enable Blakeslea strains, inparticular Blakeslea trispora, to be genetically modified and to allowproduction of homokaryotic genetically modified producer strains.

Furthermore, the method is intended to enable further carotenoids suchas, for example, xanthophylls, in particular astaxanthin or zeaxanthin,and phytoene or bixin to be produced which have previously been producedby and isolated from the wild types of the microorganisms only to a verylow extent, if at all.

It is also an object of the present invention to make available a methodfor producing carotenoids from genetically modified cells of Blakesleastrains, in particular Blakeslea trispora, which method allows the useof smaller amounts of solvent and essentially does not produce wasteand, moreover, allows high purity and higher yields.

In this connection, it is intended to utilize a very large portion ofthe nutrients present in the fermenter, both carotenoids and othernutrients present in the microorganisms.

Thus, it is also an object of the present invention to provide a methodfor producing a carotenoid-containing foodstuff which itself covers thecarotenoid requirements without additives. In particular, the nutrientcontent of the foodstuffs obtainable by said method is intended to be atleast equal to that of the previously obtainable foodstuffs. The methodis also intended to enable the produced carotenoids to be utilizedefficiently.

This object is achieved by a method for producing carotenoids or theirprecursors using genetically modified organisms of the Blakeslea genus,which method comprises the following steps:

-   -   (i) transformation of at least one of the cells,    -   (ii) optional homokaryotic conversion of the cells obtained in        step (i) to produce cells in which one or more genetic        characteristics of the nuclei are all modified in an identical        manner and said genetic modification manifests itself in the        cells, and    -   (iii) selection and reproduction of the genetically modified        cell or cells,    -   (iv) cultivation of the genetically modified cells,    -   (v) preparation of the carotenoid produced by the genetically        modified cells or the carotenoid precursor produced by said        genetically modified cells.

The method of the invention enables Blakeslea to be genetically modifiedin a specific and stable manner, in order to obtain in this way myceliumof cells with uniform nuclei, which produces carotenoids or theirprecursors, in particular xanthophylls, particularly preferablyastaxanthin or zeaxanthin, and phytoene or bixin. The cells arepreferably those of fungi of the Blakeslea trispora species. Thecarotenoids or their precursors produced here are essentially free ofcontaminations, and it is possible to achieve high concentrations ofsaid carotenoids or their precursors in the culture medium.

Transformation means the transfer of genetic information into theorganism, in particular fungus. This should include any possible methodsknown to the skilled worker of introducing said information, inparticular DNA, for example bombardment with DNA-loaded particles,transformation using protoplasts, microinjection of DNA,electroporation, conjugation or transformation of competent cells,chemicals or agrobacteria-mediated transformation. Genetic informationmeans a gene section, a gene or a plurality of genes. The geneticinformation may be introduced into the cells, for example, with the aidof a vector or as free nucleic acid (e.g. DNA, RNA) and in any othermanner, and either be incorporated into the host genome by recombinationor be present in a free form in the cell. Particular preference is givenhere to homologous recombination.

The preferred transformation method is the transformation mediated byAgrobacterium tumefaciens. To this end, the donor DNA to be transferredis first inserted into a vector which (i) carries the T-DNA endsflanking the DNA to be transferred, (ii) includes a selection marker and(iii) has, if appropriate, promoters and terminators for gene expressionof the donor DNA. Said vector is transferred into an Agrobacteriumtumefaciens strain harboring a Ti plasmid containing the vir genes. virgenes are responsible for DNA transfer in Blakeslea. This two-vectorsystem is used for transferring the DNA from Agrobacterium intoBlakeslea. To this end, the Agrobacteria are first incubated in thepresence of Acetosyringone. Acetosyringone induces the vir genes. Sporesof Blakeslea trispora are then incubated together with the induced cellsof Agrobacterium tumefaciens on Acetosyringone-containing medium andthereafter transferred to medium which enables selection of thetransformants, i.e. of the genetically modified Blakeslea strains.

The term vector is used in the present application to refer to a DNAmolecule which is used for introducing foreign DNA into and, ifappropriate, propagating said foreign DNA in a cell (see also “vector”in Römpp Lexikon Chemie—CDROM Version 2.0, Stuttgart/New York: GeorgThieme Verlag 1999). In the present application, the term “vector” isintended to include also plasmids, cosmids etc. which serve the samepurpose.

Expression means in the present application the transfer of geneticinformation, starting from DNA or RNA, to a gene product (herepreferably enzymes for producing carotenoids and in particularxanthophylls, particularly preferably astaxanthin or zeaxanthin, andphytoene or bixin), and is also intended to include the termoverexpression, meaning increased expression so as for a gene productwhich is already produced in the untransformed cell (wild type) to beincreasingly produced or to form a large part of the entire cellcontent.

Genetic modification means the introduction of genetic information intoa recipient organism so that said information is expressed in a stablemanner and passed on during cell division. In this context, homokaryoticconversion is the production of cells which contain only uniform nuclei,i.e. nuclei having the same genetic information content.

This homokaryotic conversion is only required if the genetic informationintroduced by transformation is recessive, i.e. does not manifestitself. However, if transformation results in the presence of dominantgenetic information, i.e. if said information manifests itself,homokaryotic conversion is not absolutely necessary.

The homokaryotic conversion preferably comprises selecting themononuclear spores. A small proportion of the Blakeslea trispora sporesis by nature mononuclear so that these spores can be sorted out, ifappropriate after specific labeling, for example staining, of the cellnuclei. This is preferably carried out using FACS (FluorescenceActivated Cell Sorting), on the basis of the lower fluorescence of themononuclear cells.

Alternatively, the homokaryotic conversion can be carried out by firstreducing the number of nuclei. To this end, a mutagenic agent may beemployed, in particular N-methyl-N′-nitronitrosoguanidine (MNNG). Highenergy radiation such as UV radiation or X rays may also be used forreducing the number of nuclei. The subsequent selection may be carriedout using the FACS method or recessive selection markers.

Selection means the selection of cells whose nuclei include the samegenetic information, i.e. cells which have the same properties such asresistances or production or increased production of a product.Preference is given to using for selection, aside from the FACS method,5-carbon-5-deazariboflavin (DARF) and hygromycin (hyg) or5′-fluororotate (FOA) and uracil.

The vector employed in the transformation (i) can be designed so as forthe genetic information comprised in said vector to be integrated intothe genome of at least one cell. In this connection, genetic informationin the cell may be switched off. This may be carried out directly, i.e.by way of a deletion. However, it is also possible for the vectoremployed in the transformation (i) to be designed in such a way that thegenetic information comprised in said vector is expressed in the cell,i.e. genetic information is introduced which is not present in thecorresponding wild type or which is increased or overexpressed by saidtransformation and whose product switches off the gene. The introducedgenetic information may, however, switch off genetic information in thecell also indirectly, for example by way of producing an inhibitor.

The vector employed comprises genetic information or parts of saidgenetic information for producing carotenoids or their precursors, inparticular carotenes or xanthophylls or their precursors. The vectoremployed comprises preferably genetic information for producingastaxanthin, zeaxanthin, echinenone, β-cryptoxanthin, andonixanthin,adonirubine, canthaxanthin, 3-hydroxyechinenone, 3′-hydroxyechinenone,lycopene, lutein, phytofluene, bixin or phytoene. Very particularlypreferably, the vector comprises information for producing bixin,phytoene, canthaxanthin, astaxanthin or zeaxanthin.

The vector may comprise any genetic information for geneticmodifications of organisms of the Blakeslea genus.

“Genetic information” means preferably nucleic acids whose introductioninto the organism of the Blakeslea genus results in a geneticmodification in organisms of the Blakeslea genus, i.e., for example, incausing, increasing or reducing enzyme activities in comparison with thestarting organism.

The vector may comprise, for example, genetic information for producinglipophilic substances such as, for example, carotenoids and theirprecursors, phospholipids, triacylglycerides, steroids, waxes,fat-soluble vitamins, provitamins and cofactors or genetic informationfor producing hydrophilic substances such as, for example, proteins,amino acids, nucleotides and water-soluble vitamins, provitamins andcofactors.

The vector employed preferably comprises genetic information forproducing carotenoids or xanthophylls or their precursors.

The vector preferably comprises genetic information causing thecarotenoid biosynthesis enzymes to be located in the cell compartment inwhich carotenoid biosynthesis takes place.

Particular preference is given to genetic information for producingastaxanthin, zeaxanthin, echinenone, β-cryptoxanthin, andonixanthin,adonirubin, canthaxanthin, 3- and 3′-hydroxyechinenone, lycopene,lutein, β-carotene, phytoene and/or phytofluene. Very particularpreference is given to genetic information for producing phytoene,bixin, lycopene, zeaxanthin, canthaxanthin and/or astaxanthin.

Accordingly, a preferred variant of the invention comprises producingand culturing organisms having an increased rate of synthesis ofcarotenoid biosynthesis intermediates and consequently increasedproductivity for final products of carotenoid biosynthesis. The rate ofsynthesis of carotenoid biosynthesis intermediates is increased inparticular by increasing the activities of the enzymes3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase),isopentenyl pyrophosphate isomerase and geranyl pyrophosphate synthase.

Accordingly, a particularly preferred variant of the invention comprisesproducing and culturing organisms having an increased HMG-CoA reductaseactivity compared to the wild type.

HMG-CoA reductase activity means the enzyme activity of an HMG-CoAreductase (3-hydroxy-3-methylglutaryl coenzyme A reductase).

HMG-CoA reductase means a protein which has the enzymic activity ofconverting 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate.

Accordingly, HMG-CoA reductase activity means the amount of3-hdroxy-3-methylglutaryl-coenzyme A converted or the amount ofmevalonate produced by the protein HMG-CoA reductase within a particulartime.

In the case of increased HMG-CoA reductase activity compared with thewild type, thus the protein HMG-CoA reductase increases the amount of3-hydroxy-3-methylglutaryl coenzyme A converted or the amount ofmevalonate produced within a particular time in comparison with the wildtype.

This increase in HMG-CoA reductase activity is preferably at least 5%,more preferably at least 20%, more preferably at least 50%, morepreferably at least 100%, particularly preferably at least 300%, stillmore preferably at least 500%, in particular at least 600%, of theHMG-CoA reductase activity of the wild type.

In a preferred embodiment, the HMG-CoA reductase activity is increasedcompared to the wild type by increasing gene expression of a nucleicacid encoding an HMG-CoA reductase.

In a particularly preferred embodiment of the method of the invention,gene expression of a nucleic acid encoding an HMG-CoA reductase isincreased by introducing into the organism a nucleic acid constructcomprising a nucleic acid encoding an HMG-CoA reductase whose expressionin said organism is subject to a reduced regulation, compared with thewild type.

Reduced regulation compared with the wild type means a reduced,preferably no, regulation at the expression or protein level incomparison with the wild type defined above.

Reduced regulation may preferably also be achieved by a promoter whichis functionally linked to the coding sequence in the nucleic acidconstruct and which is subject to a reduced regulation in the organism,compared with the wild type promoter.

For example, the promoters ptef1 of Blakeslea trispora and pgpdA ofAspergillus nidulans are subject only to reduced regulation and aretherefore particularly preferred promoters.

These promoters exhibit nearly constitutive expression in Blakesleatrispora so that transcriptional regulation no longer takes place viathe intermediates of carotenoid biosynthesis.

In a further preferred embodiment, said reduced regulation can beachieved by using a nucleic acid encoding an HMG-CoA reductase, whoseexpression in said organism is subject to a reduced regulation, comparedwith the orthologous nucleic acid intrinsic to said organism.

Particular preference is given to using a nucleic acid which encodesonly the catalytic region of HMG-CoA reductase (truncated (t-)HMG-CoAreductase). The membrane domain responsible for regulation is absent.The nucleic acid used is thus subject to reduced regulation and thusresults in an increase of gene expression of HMG-CoA reductase.

In a particularly preferred embodiment, nucleic acids comprising thesequence SEQ ID. NO. 75 are introduced into Blakeslea trispora.

Further examples of HMG-CoA reductases and thus also of the t-HMG-CoAreductases reduced to the catalytic region or the encoding genes canreadily be found, for example, from various organisms whose genomicsequence is known by homology comparisons of the sequences fromdatabases with SEQ ID. NO. 75.

Further examples of HMG-CoA reductases and thus also of the t-HMG-CoAreductases reduced to the catalytic region or the encoding genes canfurthermore readily be found, for example starting from the sequence SEQID. NO. 75, from various organisms whose genomic sequence is not known,by hybridization and PCR techniques in a manner known per se.

In a particularly preferred embodiment, said reduced regulation isachieved by using a nucleic acid encoding an HMG-CoA reductase, whoseexpression in said organism is subject to a reduced regulation, comparedwith the orthologous nucleic acid intrinsic to said organism, and usinga promoter which is subject to a reduced regulation in said organism,compared with the wild type promoter.

Accordingly, a preferred variant of the invention comprises thetransformation switching off phytoene desaturase gene expression, thusenabling the phytoene produced by the organisms to be isolated. Thevector employed in the transformation (i) therefore comprises in oneembodiment of the invention preferably a sequence coding for a fragmentof the gene of phytoene desaturase, in particular Blakeslea trisporacarB, with SEQ ID NO: 69.

Accordingly, a preferred variant of the invention comprises lycopenecyclase gene expression being switched off by transformation, thusenabling the lycopene produced by the organisms to be isolated. Thevector employed in said transformation therefore comprises in oneembodiment of the invention preferably a sequence coding for a fragmentof the lycopene cyclase gene, in particular Blakeslea trispora carR.

In a preferred embodiment, the organisms of the Blakeslea genus areenabled, for example, to produce xanthophylls such as, for example,canthaxanthin, zeaxanthin or astaxanthin, bixin or phytoene by causing ahydroxylase activity and/or ketolase activity in the geneticallymodified organisms of the Blakeslea genus, in comparison with the wildtype.

Thus, in a further, preferred variant of the invention, the vectoremployed in the transformation (i) comprises genetic information which,after expression, displays a ketolase and/or hydroxylase activity sothat the organisms produce zeaxanthin or astaxanthin.

Ketolase activity means the enzyme activity of a ketolase.

A ketolase means a protein which has the enzymic activity of introducinga keto group at the optionally substituted β-ionone ring of carotenoids.

A ketolase means in particular a protein which has the enzymic activityof converting β-carotene to canthaxanthin.

Accordingly, ketolase activity means the amount of β-carotene convertedor the amount of canthaxanthin produced by the protein ketolase within aparticular time.

According to the invention, the term “wild type” means the correspondinggenetically unmodified starting organism of the Blakeslea genus.

The term “organism” may mean the starting organism (wild type) of theBlakeslea genus or a genetically modified organism according to theinvention of the Blakeslea genus or both, depending on the context.

Preferably “wild type” for causing the ketolase activity and for causingthe hydroxylase activity means in each case a reference organism.

This reference organism of the Blakeslea genus is Blakeslea trisporaATCC 14271 or ATCC 14272 which differ merely with respect to the matingtype.

The ketolase activity in genetically modified organisms according to theinvention of the Blakeslea genus and in wild type or reference organismsis preferably determined under the following conditions:

The ketolase activity in organisms of the Blakeslea genus is determinedfollowing the method of Frazer et al., (J. Biol. Chem. 272(10):6128-6135, 1997). The ketolase activity in extracts is determined usingthe substrates beta-carotene and canthaxanthin in the presence of lipid(soya lecithin) and detergent (sodium cholate). Substrate-to-productratios of the ketolase assays are determined by means of HPLC.

In this preferred embodiment, the genetically modified organismaccording to the invention of the Blakeslea genus has, in comparisonwith the genetically unmodified wild type, a ketolase activity and isthus preferably capable of transgenically expressing a ketolase.

In a further preferred embodiment, the ketolase activity in theorganisms of the Blakeslea genus is caused by causing gene expression ofa nucleic acid encoding a ketolase.

In this preferred embodiment, gene expression of a nucleic acid encodinga ketolase is preferably caused by introducing nucleic acids encodingketolases into the starting organism of the Blakeslea genus.

For this purpose, it is possible in principle to use any ketolase gene,i.e. any nucleic acid encoding a ketolase.

Any of the nucleic acids mentioned in the description may be an RNA, DNAor cDNA sequence for example.

In the case of genomic ketolase sequences from eukaryotic sources, whichinclude introns, preference is given to using already processed nucleicacid sequences such as the corresponding cDNAs, if the host organism ofthe Blakeslea genus is unable or cannot be made to express thecorresponding ketolase.

Examples of nucleic acids encoding a ketolase and the correspondingketolases, which may be used in the method of the invention, are, forexample, sequences from:

Haematoccus pluvialis, in particular from Haematoccus pluvialis Flotowem. Wille (accession NO: X86782; nucleic acid: SEQ ID NO: 11, proteinSEQ ID NO: 12),

Haematoccus pluvialis, NIES-144 (accession NO: D45881; nucleic acid: SEQID NO: 13, protein SEQ ID NO: 14),

Agrobacterium aurantiacum (accession NO: D58420; nucleic acid: SEQ IDNO: 15, protein SEQ ID NO: 16),

Alicaligenes spec. (accession NO: D58422; nucleic acid: SEQ ID NO: 17,protein SEQ ID NO: 18),

Paracoccus marcusii (accession NO: Y15112; nucleic acid: SEQ ID NO: 19,protein SEQ ID NO: 20).

Synechocystis sp. Strain PC6803 (accession NO: NP442491; nucleic acid:SEQ ID NO: 21, protein SEQ ID NO: 22).

Bradyrhizobium sp. (accession NO: AF218415; nucleic acid: SEQ ID NO: 23,protein SEQ ID NO: 24).

Nostoc sp. Strain PCC7120 (accession NO: AP003592, BAB74888; nucleicacid: SEQ ID NO: 25, protein SEQ ID NO: 26),

Nostoc punctiforme ATTC 29133, Nucleic acid: Acc. No. NZ_AABC01000195,base pair 55,604 to 55,392 (SEQ ID NO: 27); Protein: Acc. No.ZP_(—)00111258 (SEQ ID NO: 28) (annotated as putative protein),

Nostoc punctiforme ATTC 29133, Nucleic acid: Acc. No. NZ_AABC01000196,base pair 140,571 to 139,810 (SEQ ID NO: 29), protein: (SEQ ID NO: 30)(not annotated),

Further natural examples of ketolases and ketolase genes, which may beused in the process of the invention, can be readily found, for example,from various organisms whose genomic sequence is known by comparing theidentities of the amino acid sequences or of the corresponding backtranslated nucleic acid sequences from databases with those of thepreviously described sequences and in particular with those of thesequences SEQ ID NO: 12, 26 and/or 33.

Further natural examples of ketolases and ketolase genes can furthermorebe readily found, starting from the previously described nucleic acidsequences, in particular starting from the sequences SEQ ID NO: 12, 26and/or 30, from various organisms whose genomic sequence is not known,using hybridization techniques in a manner known per se.

The hybridization may be carried out under moderate (low stringency) or,preferably, under stringent (high stringency) conditions.

Hybridization conditions of these types are described, for example, inSambrook, J., Fritsch, E. F., Maniatis, T., in: Molecular Cloning (ALaboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press,1989, pages 9.31-9.57 or in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

For example, the conditions during the washing step may be selected fromthe range of conditions limited by those of low stringency (with 2×SSCat 50° C.) and those of high stringency (with 0.2×SSC at 50° C.,preferably at 65° C.) (20×SSC: 0.3 M sodium citrate, 3 M sodiumchloride, pH 7.0).

An additional possibility is to rise the temperature during the washingstep from moderate conditions at room temperature, 22° C., up tostringent conditions at 65° C.

Both parameters, the salt concentration and temperature, can be variedsimultaneously, and it is also possible to keep one of the twoparameters constant and vary only the other one. It is also possible toemploy denaturing agents such as, for example, formamide or SDS duringthe hybridization. Hybridization in the presence of 50% formamide ispreferably carried out at 42° C.

Some examples of conditions for hybridization and washing step are givenbelow:

(1) hybridization conditions with, for example,

(i) 4×SSC at 65° C., or

(ii) 6×SSC at 45° C., or

(iii) 6×SSC at 68° C., 100 mg/ml denatured fish sperm DNA, or

(iv) 6×SSC, 0.5% SDS, 100 mg/ml denatured, fragmented salmon sperm DNAat 68° C., or

(v) 6×SSC, 0.5% SDS, 100 mg/ml denatured, fragmented salmon sperm DNA,50% formamide at 42° C., or

(vi) 50% formamide, 4×SSC at 42° C., or (vii) 50% (vol/vol) formamide,0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mMsodium phosphate buffer pH 6.5, 750 mM NaCl, 75 mM sodium citrate at 42°C., or

(viii) 2× or 4×SSC at 50° C. (moderate conditions), or

(ix) 30 to 40% formamide, 2× or 4×SSC at 42° C. (moderate conditions).

(2) Washing steps of 10 minutes each with, for example,

(i) 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C., or

(ii) 0.1×SSC at 65° C., or

(iii) 0.1×SSC, 0.5% SDS at 68° C., or

(iv) 0.1×SSC, 0.5% SDS, 50% formamide at 42° C., or

(v) 0.2×SSC, 0.1% SDS at 42° C., or

(vi) 2×SSC at 65° C. (moderate conditions).

In a preferred embodiment of the genetically modified organismsaccording to the invention of the Blakeslea genus, nucleic acids areintroduced which encode a protein comprising the amino acid sequence SEQID NO: 12 or a sequence which is derived from this sequence bysubstitution, insertion or deletion of amino acids and which has anidentity of at least 20%, preferentially at least 30%, 40%, 50%, 60%,preferably at least 70%, 80%, particularly preferably at least 90%, inparticular 91%, 92%; 93%, 94%, 95%, 96%, 97%, 98% or 99%, at the aminoacid level with the sequence SEQ ID NO: 12 and which has the enzymicproperty of a ketolase.

In this connection, it is possible for the ketolase sequence to be anatural one which can be found as described above by identity comparisonof the sequences from other organisms, or for the ketolase sequence tobe an artificial one which has been modified starting from the sequenceSEQ ID NO: 12 by artificial variation, for example by substitution,insertion or deletion of amino acids.

A further, preferred embodiment of the methods of the invention involvesintroducing nucleic acids which encode a protein comprising the aminoacid sequence SEQ ID NO: 26 or a sequence which is derived from thissequence by substitution, insertion or deletion of amino acids and whichhas an identity of at least 20%, preferentially at least 30%, 40%, 50%,60%, preferably at least 70%, 80%, particularly preferably at least 90%,in particular 91%, 92%; 93%, 94%, 95%, 96%, 97%, 98% or 99%, at theamino acid level with the sequence SEQ ID NO: 26 and which has theenzymic property of a ketolase.

In this connection, it is possible for the ketolase sequence to be anatural one which can be found as described above by identity comparisonof the sequences from other organisms, or for the ketolase sequence tobe an artificial one which has been modified starting from the sequenceSEQ ID NO: 26 by artificial variation, for example by substitution,insertion or deletion of amino acids.

A further, preferred embodiment of the methods of the invention involvesintroducing nucleic acids which encode a protein comprising the aminoacid sequence SEQ ID NO: 30 or a sequence which is derived from thissequence by substitution, insertion or deletion of amino acids and whichhas an identity of at least 20%, preferentially at least 30%, 40%, 50%,preferably at least 60%, 70%, more preferably at least 80%, 85%,particularly preferably at least 90%, in particular 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99%, at the amino acid level with the sequence SEQID NO: 30 and which has the enzymic property of a ketolase.

In this connection, it is possible for the ketolase sequence to be anatural one which can be found as described above by identity comparisonof the sequences from other organisms, or for the ketolase sequence tobe an artificial one which has been modified starting from the sequenceSEQ ID NO: 30 by artificial variation, for example by substitution,insertion or deletion of amino acids.

The term “substitution” means in the description substitution of one ormore amino acids by one or more amino acids. Preference is given tocarrying out “conservative” substitutions in which the replaced aminoacid has a similar property to the original amino acid, for examplesubstitution of Glu by Asp, Gln by Asn, Val by Ile, Leu by Ile, Ser byThr.

Deletion is the replacement of an amino acid by a direct bond. Preferredpositions for deletions are the termini of the polypeptide and thelinkages between the individual protein domains.

Insertions are insertions of amino acids into the polypeptide chain,with formal replacement of a direct bond by one or more amino acids.

Identity between two proteins means the identity of the amino acids overthe entire length of each protein, in particular the identity calculatedby comparison with the aid of Lasergene software from DNASTAR, inc.Madison, Wis. (USA) using the Clustal method (Higgins D G, Sharp P M.Fast and sensitive multiple sequence alignments on a microcomputer.Comput Appl. Biosci. 1989 April; 5(2):151-1), setting the followingparameters:

Multiple Alignment Parameter: Multiple alignment parameter: Gap penalty10 Gap length penalty 10 Pairwise alignment parameter: K-tuple 1 Gappenalty 3 Window 5 Diagonals saved 5

Accordingly, a protein which has an identity of at least 20% at theamino acid level with the sequence SEQ ID NO: 12 or 26 or 30 means aprotein which, on comparison of its sequence with the sequence SEQ IDNO: 12 or 26 or 30, in particular using the above program logarithm withthe above set of parameters, has an identity of at least 20%, preferably30%, 40%, 50%, particularly preferably 60%, 70%, 80%, in particular 85%,90, 95%.

Suitable nucleic acid sequences can be obtained, for example, by backtranslation of the polypeptide sequence in accordance with the geneticcode.

The codons preferably used for this purpose are those frequently usedaccording to the Blakeslea-specific codon usage. The codon usage caneasily be found by means of computer analyses of other, known genes oforganisms of the Blakeslea genus.

In a particularly preferred embodiment, a nucleic acid comprising thesequence SEQ ID NO: 11 is introduced into the organism of said genus.

In a particularly preferred embodiment, a nucleic acid comprising thesequence SEQ ID NO: 25 is introduced into the organism of said genus.

In a particularly preferred embodiment, a nucleic acid comprising thesequence SEQ ID NO: 29 is introduced into the organism of said genus.

All the aforementioned ketolase genes can moreover be prepared in amanner known per se by chemical synthesis from the nucleotide buildingblocks, for example by fragment condensation of individual overlapping,complementary nucleic acid building blocks of the double helix. Chemicalsynthesis of oligonucleotides is possible, for example, in a knownmanner by the phosphoamidite method (Voet, Voet, 2nd edition, WileyPress New York, pages 896-897). Addition of synthetic oligonucleotidesand filling in of gaps with the aid of the Klenow fragment of DNApolymerase and ligation reactions, and also general cloning methods aredescribed in Sambrook et al. (1989), Molecular cloning: A laboratorymanual, Cold Spring Harbor Laboratory Press.

The vector employed in the transformation (i) therefore comprises in oneembodiment of the invention preferably a sequence coding for a ketolase,in particular the Nostoc punctiforme ketolase with SEQ ID NO: 72.

Hydroxylase activity means the enzymic activity of a hydroxylase.

A hydroxylase means a protein having the enzymic activity of introducinga hydroxyl group on the, optionally substituted, β-ionone ring ofcarotenoids.

In particular, a hydroxylase means a protein having the enzymic activityof converting β-carotene to zeaxanthin or cantaxanthin to astaxanthin.

Accordingly, hydroxylase activity means the amount of β-carotene orcantaxanthin converted, or amount of zeaxanthin or astaxanthin produced,by the hydroxylase protein in a particular time.

Thus, when the hydroxylase activity is increased compared with the wildtype, the amount of β-carotene or cantaxantin converted or the amount ofzeaxanthin or astaxanthin produced in a particular time by thehydroxylase protein is increased in comparison with the wild type.

This increase in hydroxylase activity is preferably at least 5%, furtherpreferably at least 20%, further preferably at least 50%, furtherpreferably at least 100%, more preferably at least 300%, still morepreferably at least 500%, in particular at least 600%, of thehydroxylase activity of the wild type.

The hydroxylase activity in the genetically modified organisms of theinvention and in wild-type and reference organisms is preferablydetermined under the following conditions:

The hydroxylase activity is determined by the method of Bouvier et al.(Biochim. Biophys. Acta 1391 (1998), 320-328) in vitro. Ferredoxin,Ferredoxin-NADP oxidoreductase, katalase, NADPH and beta-carotene areadded with mono- and digalactosyl glycerides to a defined amount oforganism extract.

The hydroxylase activity is particularly preferably determined under thefollowing conditions of Bouvier, Keller, d'Harlingue and Camara(Xanthophyll biosynthesis: molecular and functional characterization ofcarotenoid hydroxylases from pepper fruits (Capsicum annuum L.; Biochim.Biophys. Acta 1391 (1998), 320-328):

The in vitro assay is carried out in a volume of 0.250 ml. The mixturecontains 50 mM potassium phosphate (pH 7.6), 0.025 mg of spinachferredoxin, 0.5 unit of spinach ferredoxin-NADP+ oxidoreductase, 0.25 mMNADPH, 0.010 mg of beta-carotene (emulsified in 0.1 mg of Tween 80),0.05 mM of a mixture of mono- and digalactosyl glycerides (1:1), 1 unitof catalysis, 200 mono- and digalactosyl glycerides, (1:1), 0.2 mg ofbovine serum albumin and organism extract in a varying volume. Thereaction mixture is incubated at 30° C. for 2 hours. The reactionproducts are extracted with an organic solvent such as acetone orchloroform/methanol (2:1) and determined by HPLC.

The hydroxylase activity is particularly preferably determined under thefollowing conditions of Bouvier, d'Harlingue and Camara (MolecularAnalysis of carotenoid cyclae inhibition; Arch. Biochem. Biophys. 346(1)(1997) 53-64):

The in vitro assay is carried out in a volume of 250 μl. The mixturecontains 50 mM potassium phosphate (pH 7.6), varying amounts of organismextract, 20 nM lycopene, 250 μg of paprika chromoplastid stromalprotein, 0.2 mM NADP+, 0.2 mM NADPH and 1 mM ATP. NADP/NADPH and ATP aredissolved in 10 ml of ethanol with 1 mg of Tween 80 immediately beforeaddition to the incubation medium. After a reaction time of 60 minutesat 30° C., the reaction is stopped by adding chloroform/methanol (2:1).The reaction products extracted into chloroform are analyzed by HPLC.

An alternative assay with radioactive substrate is described in Fraserand Sandmann (Biochem. Biophys. Res. Comm. 185(1) (1992) 9-15).

The hydroxylase activity can be increased in various ways, for exampleby switching off inhibitory regulatory mechanisms at the expression andprotein levels or by increasing gene expression of nucleic acidsencoding a hydroxylase, compared with the wild type.

Gene expression of the nucleic acids encoding a hydroxylase can likewisebe increased, compared with the wild type, in various ways, for exampleby inducing the hydroxylase gene by activators or by introducing one ormore hydroxylase gene copies, i.e. by introducing at least one nucleicacid encoding a hydroxylase into the organism of the Blakeslea genus.

In a preferred embodiment, gene expression of a nucleic acid encoding ahydroxylase is increased by introducing at least one nucleic acidencoding a hydroxylase into the organism of the Blakeslea genus.

It is possible to use for this purpose in principle any hydroxylasegene, i.e. any nucleic acid which encodes a hydroxylase and any nucleicacid which encodes a cyclase.

In the case of genomic hydroxylase sequences from eukaryotic sources,which comprise introns, preference is given to using nucleic acidsequences which have already been processed, such as the correspondingcDNAs, if the host organism is unable or cannot be made to express thecorresponding hydroxylase.

One example of a hydroxylase gene is a nucleic acid encoding aHaematococcus pluvialis hydroxylase, with accession No. AX038729 (WO0061764; nucleic acid: SEQ ID NO: 31, protein: SEQ ID NO: 32), anErwinia uredovora 20D3 hydroxylase (ATCC 19321, accession No. D90087;nucleic acid: SEQ ID NO: 33, protein: SEQ ID NO: 34) or Thermusthermophilus hydroxylase (DE 102 34 126.5) encoded by the sequence SEQID NO 76.

and also hydroxylases of the following accession numbers:|emb|CAB55626.1, CAA70427.1, CAA70888.1, CAB55625.1, AF499108_(—)1,AF315289_(—)1, AF296158_(—)1, AAC49443.1, NP_(—)194300.1,NP_(—)200070.1, AAG10430.1, CAC06712.1, AAM88619.1, CAC95130.1,AAL80006.1, AF162276_(—)1, AA053295.1, AAN85601.1, CRTZ_ERWHE,CRTZ_PANAN, BAB79605.1, CRTZ_ALCSP, CRTZ_AGRAU, CAB56060.1,ZP_(—)00094836.1, AAC44852.1, BAC77670.1, NP_(—)745389.1,NP_(—)344225.1, NP_(—)849490.1, ZP_(—)00087019.1, NP_(—)503072.1,NP_(—)852012.1, NP_(—)115929.1, ZP_(—)00013255.1

Thus, in this preferred embodiment, at least one further hydroxylasegene is present in the preferred transgenic organisms according to theinvention of the Blakeslea genus, compared with the wild type.

In this preferred embodiment, the genetically modified organism has, forexample, at least one exogenous nucleic acid encoding a hydroxylase orat least two endogenous nucleic acids encoding a hydroxylase.

In the preferred embodiment described above, preference is given tousing as hydroxylase genes nucleic acids which encode proteinscomprising the amino acid sequence SEQ ID NO: 32, 34 or encoded by thesequence SEQ ID NO 76 or a sequence which is derived from this sequenceby substitution, insertion or deletion of amino acids and which has anidentity of least 30%, preferably at least 50%, more preferably at least70%, still more preferably at least 80%, more preferably at least 90%,in particular 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, at the aminoacid level to the sequence SEQ. ID. NO: 32, 34, or encoded by thesequence with SEQ ID NO 76, and which have the enzymic property of ahydroxylase.

Further examples of hydroxylases and hydroxylase genes can readily befound, for example, from various organisms whose genomic sequence isknown, as described above, by homology comparisons of the amino acidsequences or of the corresponding back-translated nucleic acid sequencesfrom databases with SEQ ID. NO: 31, 33 or 76.

Further examples of hydroxylases and hydroxylase genes can furthermorereadily be found in a manner known per se, for example starting from thesequence SEQ ID NO: 31, 33 or 76, from various organisms whose genomicsequence is unknown, as described above, by hybridization and PCRtechniques.

In a further particularly preferred embodiment, nucleic acids whichencode proteins comprising the amino acid sequence of the hydroxylase ofsequence SEQ ID NO: 32, 34 or encoded by the sequence SEQ ID NO 76 areintroduced into organisms to increase the hydroxylase activity.

Suitable nucleic acid sequences can be obtained, for example, by backtranslation of the polypeptide sequence in accordance with the geneticcode.

Preference is given to using for this purpose those codons which arefrequently used in accordance with the organism-specific codon usage.The codon usage can readily be determined on the basis of computeranalyses of other, known genes of the organisms in question.

In a particularly preferred embodiment, a nucleic acid comprising thesequence SEQ. ID. NO: 31, 33 or 76 is introduced into the organism.

All the aforementioned hydroxylase genes can furthermore be prepared ina manner known per se by chemical synthesis from the nucleotide buildingblocks, for example by fragment condensation of individual overlapping,complementary nucleic acid building blocks of the double helix. Chemicalsynthesis of oligonucleotides is possible, for example, in a knownmanner by the phosphoamidite method (Voet, Voet, 2nd edition, WileyPress New York, pages 896-897). Addition of synthetic oligonucleotidesand filling in of gaps with the aid of the Klenow fragment of DNApolymerase and ligation reactions, and also general cloning methods aredescribed in Sambrook et al. (1989), Molecular cloning: A laboratorymanual, Cold Spring Harbor Laboratory Press.

The vector employed in the transformation (i) therefore comprises in afurther embodiment of the invention preferably a sequence coding for ahydroxylase, in particular a Haematococcus pluvialis hydroxylase withSEQ ID NO: 70 or an Erwinia uredova hydroxylase with SEQ ID NO: 71 or aThermus thermophilus hydroxylase encoded by the sequence SEQ ID NO 76.

Preference is given to the transformation switching off the gene ofphytoene desaturase.

The vector employed in the transformation (i) preferably also includesregions which control and support expression, in particular promotersand terminators.

The vector employed in the transformation (i) preferably includes thegpd and/or the ptef1 promoter and/or the trpc terminator, all of whichhave proved to be particularly successful in the transformation ofBlakeslea. The use of “inverted repeats” familiar to the skilled worker(IR, Römpp Lexikon der Biotechnologie 1992, Thieme Verlag Stuttgart,page 407 “Inverse repetitive sequences”) for controlling expression andtranscription is also within the scope of the invention.

The gpd promoter employed in the vector has advantageously the sequenceSEQ ID NO: 1. The trpc terminator employed in the vector hasadvantageously the sequence SEQ ID NO: 2. The ptef1 promoter employed inthe vector has advantageously the sequence SEQ ID NO: 35.

Preference is given here to using in particular the gpd promoter and thetrpC terminator from Aspergillus nidulans and the ptef1 promoter fromBlakeslea trispora.

The vector employed in the transformation (i) in particular comprises aresistance gene. The latter is preferably a hygromycin resistance gene(hph), in particular one from E. coli. This resistance gene has provedparticularly suitable in the detection of transformation and selectionof the cells.

The preferred promoter utilized for hph thus is p-gpdA, the promoter ofglyceraldehyde 3-phosphate dehydrogenase coding for Aspergillusnidulans. The preferred terminator utilized for hph is t-trpC, theterminator of the trpC gene coding for Aspergillus nidulans anthranilatesynthase components.

Derivatives of the pBinAHyg vector have proved to be particularlysuitable vectors. The vector employed for transformation thus preferablycomprises SEQ ID NO: 3. To this will be added, depending on the desiredcarotenoid or its precursor, a sequence coding for a hydroxylase,ketolase, phytoene desaturase etc., as described above. The vectors thuscomprise in one embodiment of the invention the sequence SEQ ID NO: 69coding for said phytoene desaturase. The vectors also comprise in afurther embodiment of the invention the sequence SEQ ID NO: 72 codingfor a ketolase. The vectors further comprise in a further embodiment ofthe invention the sequence SEQ ID NO: 70 or 71 or 76 coding for ahydroxylase. Corresponding combinations of the abovementioned sequencesare also within the scope of the invention. Thus, the vector comprisesin one embodiment both a sequence SEQ ID NO: 72 coding for a ketolaseand the sequence SEQ ID NO: 70 or 71 or 76 coding for a hydoxylase andthus enables astaxanthin to be produced.

In particular, it is possible to use within the scope of the inventionvectors selected from the group consisting of SEQ ID NO: 37 to 51 and62.

The genetically modified organisms may be used for producingcarotenoids, xanthophylls or their precursors, in particular bixin,phytoene, astaxanthin, zeaxanthin and canthaxanthin. It is alsopossible, by introducing the appropriate genetic information, for newcarotenoids which do not occur naturally in the wild type to begenerated by the specifically genetically modified cells or by themycelium formed thereby and subsequently to be isolated.

After selection, the genetically modified cells are cultured in order tobe able to provide carotenoids or their precursors.

Preference is given to obtaining carotenoids or their precursors usingthe specifically genetically modified cells or the mycelium formedthereby.

The cultivation of the organisms has no special requirements.Advantageously, in particular when using Blakeslea trispora, oppositemating types are cultured together, since this results in better growthand production.

If the genetic modification is carried out only in cells of one of themating types found ((+) or (−) for Blakeslea trispora), thecorresponding other, unmodified mating type is added to the cultivation,since it is possible in this way to achieve good production of thecarotenoids or their precursors, owing to the substances released by thesecond, unmodified mating type (e.g. trisporic acids). Advantageously,however, the genetic modification is carried out in cells of both matingtypes which are then cultured together, thereby achieving particularlygood growth and optimal production of the carotenoids or theirprecursors. An (artificial) addition of trisporic acids is possible anduseful.

Trisporic acids are sex hormones in Mucorales fungi such as Blakeslea,which stimulate the formation of zygophores and production of β-carotene(van den Ende 1968, J. Bacteriol. 96:1298-1303, Austin et al. 1969,Nature 223:1178-1179, Reschke Tetrahedron Lett. 29:3435-3439, van denEnde 1970, J. Bacteriol. 101:423-428).

It is possible to use any media familiar to the skilled worker, as longas they are suitable for culturing the organisms used and carotenoidproduction thereof. In particular, the use of carotenoid biosynthesisinhibitors is not necessary when the “GMO” (genetically modifiedorganisms) are used. The media employed preferably include additivessuch as one or more carbon sources, one or more nitrogen sources,mineral salts and thiamine. Preference is given to employing additivesas disclosed in WO 03/038064 A2, page 4, line 30 to page 5 line 7. Aparticularly preferred carbon source is glucose and particularlypreferred nitrogen sources are asparagine, plant or animal extracts suchas cotton seed oil, soybean oil, cotton seed meal or yeast extract.

The cultivation may be carried out either under aerobic or anaerobicconditions. A mixed, first aerobic and then anaerobic, cultivation, asdisclosed in DE 101 30 323, is also possible. In this case, temperatureand humidity are set in each case for optimal growth. The temperature ofthe cultivation is preferably between approx. 20 and approx. 34° C., inparticular between approx. 26° C. and approx. 28° C. Furthermore, thecultivation may be carried out continuously or batchwise.

The cultivation is preferably carried out up to a solids content betweenabout 1 and about 20%, preferably 3 and 15% and particularly preferably4 and 11%. Particularly important is the fact that the culture brothremains pumpable so as to remain processible in the subsequent processsteps. If the solids content is too low, complicated concentration ordrying steps are required.

The cultivation or fermentation may be carried out in the usualapparatus. This includes all apparatus suitable for the microorganismsemployed in each case and their products, in particular those indicatedunder the keyword “bioreactor” on pages 123-126 of Römpp LexikonBiotechnologie (1992 Georg Thieme Verlag, Stuttgart). Particularpreference is given to using stirred tank reactors with various internalfittings, various types of bubble columns, etc.

The carotenoids or their precursors provided by the method of theinvention, in particular bixin, phytoene or xanthophylls, particularlypreferably astaxanthin or zeaxanthin, are particularly suitable forproducing additives for feedstuffs, foodstuffs and food supplements,cosmetic, pharmaceutical or dermatological preparations.

The carotenoid produced by the genetically modified cells or thecarotenoid precursor produced by the genetically modified cells isprepared from the culture of the genetically modified microorganismsaccording to two variants, a) or b), with preference also being given toa combination of a) and b);

a:

-   -   I) removal of the biomass,        -   IA) optional washing of the biomass with a solvent in which            carotenoids are not soluble, in particular water,        -   IB) sterilization and cell disruption of the biomass,        -   IC) optional drying and/or homogeneous distribution, and    -   II) partial extraction of the carotenoids from the disrupted        biomass by means of a carotenoid-dissolving solvent and        separation of said solvent from said biomass,        -   IIA)        -   1) removal of residual solvent from the            carotenoid-containing biomass,        -   2) optional homogeneous suspension of the biomass, with a            biomass solid content of >2% and <50%, and        -   3) drying of the biomass or suspension for producing the            foodstuff,        -   IIB)        -   1) crystallization of the carotenoids from the solvent used            and isolation of the carotenoid crystals, in particular by            filtration;            or b):    -   I) homogeneous suspension of the solids of the culture broth,        and    -   IIA) for a solid content of the culture broth of >2%:        -   1) optional concentration of the culture broth to give a            solid content of <50%, and        -   2) drying of the culture broth to produce the foodstuff, or    -   IIB) for a solid content of <2% of the culture broth,        -   1) concentration of the culture broth to give a solid            content of >2% and <50%, and        -   2) drying of the suspension to produce the foodstuff, or    -   IIC) independently of the solid content of the culture broth,        -   1) removal of the biomass,        -   2) optional washing of the biomass with solvents in which            carotenoids are not soluble, in particular water,        -   3) sterilization and cell disruption,        -   4) optional drying and homogeneous distribution,        -   5) partial extraction of the carotenoids from the biomass            using a carotenoid-dissolving solvent,        -   5a) removal of the carotenoid-containing biomass from the            carotenoid-containing solvent,        -   5b) removal of residual solvent from the biomass, and        -   5c) drying of the biomass to produce the foodstuff,        -   6) crystallization of the carotenoids from the solvent used            in 5a) and isolation of the carotenoid crystals, in            particular by filtration.

The preparation according to the invention of the carotenoid produced bythe genetically modified cells or of the carotenoid precursor producedby the genetically modified cells from the culture of the geneticallymodified microorganisms, carried out according to two variants a) or b),enables two products to be produced at the same time.

By combining according to the invention the production of two products,in particular in the preparation according to variant a), namely the atleast one carotenoid and the carotenoid-containing foodstuff, there isno need for complete extraction of the carotenoids from the biomass sothat said extraction is less complicated. Despite complete utilization,the carotenoid needs to be extracted only partially, and no product islost. This requires small amounts of solvent, accompanied by fewermeasures for their reuse. Moreover, waste products are largely avoided,since the biomass does not end up as waste but is processed further togive a foodstuff of high value. As a result, the methods become lessexpensive due to the utilization of synergies.

Thus, foodstuffs obtainable by the method according to the inventionwith preparation according to variant b) include already afterproduction large amounts of carotenoids which need not be added.Moreover, the nutrient content of said foodstuff is increased, due tothe fact that it also contains Blakeslea trispora, in addition to the atleast one carotenoid. The increase in nutrient content is particularlylarge according to the preferred alternatives IIA and IIB, since itincludes, aside from the at least one carotenoid and Blakeslea trispora,in addition all media components of the fermentation. Furthermore, theprocess does not require any additional, complex work-up and preparationsteps; rather, the homogenized and, if appropriate, dehydrated culturebroth containing Blakeslea trispora can be dried directly to produce thefoodstuff. As a result, there are virtually no waste products, apartfrom the aqueous medium in alternative IIB, which, however, can bepurified without problems in a purification plant. In addition, allthree alternatives utilize the entire amount of carotenoids producedwithout or with only marginal losses, since, according to IIA and IIB,no separation or work-up steps with heavy losses need to be carried out.In alternative IIIC, the entire amount of carotenoids produced islikewise utilized without or with only marginal losses, since one partis processed within the biomass to give the foodstuff and the other partis extracted to obtain pure carotenoids. The combination according tothe invention of producing two products according to IIC, namely thecarotenoid-containing foodstuff and the carotenoids per se, results inthe advantage that again essentially no waste products occur andcomplete extraction of the carotenoids from the biomass is unnecessaryso that the usually complex extraction is less complex. Despite completeutilization, the valuable carotenoid(s) need to be extracted onlypartially, without incurring product losses.

This requires small amounts of solvent, accompanied by fewer measuresfor their reuse. Moreover, waste products are largely avoided, since thebiomass does not end up as waste but is processed further to give afoodstuff of high value. As a result, the methods become less expensivedue to the utilization of synergies.

In the present application, “highly pure” means a purity of the at leastone carotenoid of at least 95%, preferably >95%, preferentially >96%,particularly preferably >97%, very particularly preferably >98%, mostpreferably >99%.

Suitable carotenoids which can be produced by the method of theinvention are all natural and artificial carotenes and xanthophylls. Theat least one carotenoid is in particular selected from the groupconsisting of astaxanthin, zeaxanthin, echinenone, β-cryptoxanthin,andonixanthin, adonirubin, canthaxanthin, 3-hydroxyechinenone,3′-hydroxyechinenone, lycopene, β-carotene, lutein, phytofluene, bixinand phytoene. Preference is given here to astaxanthin or zeaxanthin. Thecarotenoids may be obtained by the method of the invention-individuallyor as mixtures of two or more of the abovementioned carotenoids. Thecarotenoid or carotenoids may be produced specifically, in particularwhen using the genetically modified organisms (GMO) indicatedhereinbelow.

Foodstuffs are regarded as compositions used for nutrition. These alsoinclude compositions for supplementing nutrition. Animal feedstuffs andanimal feed supplements, in particular, are regarded as foodstuffs.

After cultivation, the biomass can be removed from the culture brothaccording to variant a) of the preparation. To this end, any methods ofsolid/liquid separation familiar to and usually employable by theskilled worker may be used. These include in particular the mechanicalprocesses, such as filtration and centrifugation, which are based onutilizing gravity, centrifugal force, pressure or vacuum. The processesand apparatus which may be used include in addition, inter alia, crossflow filtration or membrane techniques such as osmosis, reverse osmosis,microfiltration, ultrafiltration, nanofiltration, cake filtrationprocesses (e.g. by means of automatic pressure filters (membrane, frameor chamber) filter presses, (agitated) pressure filters, suctionfilters, (vacuum) belt filters, (vacuum) drum filters, rotary filters,candle filters), centrifugation processes by means of continuously orbatchwise operated centrifuges or filter centrifuges (e.g. invertingfilter centrifuges, scraper centrifuges, pusher-type centrifuges,worm/screen centrifuges, slide centrifuges, separators or decantercentrifuges), processes utilizing gravity, such as flotation,sedimentation, sink-float purification and clarifying. The biomass isremoved from the culture broth preferably by centrifugation by means ofa decanter or by filtration by means of a membrane filtration unit.

The second step of the preparation according to variant b) generates ahomogeneously distributed suspension of the solids in the culture broth.To this end, any methods familiar to and usually employable by theskilled worker may be used. Use is made here (on the laboratory scale)in particular of dispersers such as an UltraTurrax®. Cell disruption maybe carried out but is not necessary.

The culture broth may, if necessary, be dehydrated in order to achieve asuitable solid content of between >2% and <50%. To this end, any methodsof solid/liquid separation familiar to and usually employable by theskilled worker may be used. These include in particular the mechanicalprocesses, such as filtration and centrifugation, which are based onutilizing gravity, centrifugal force, pressure or vacuum. The processesand apparatus which may be used include in addition, inter alia, crossflow filtration or membrane techniques such as osmosis, reverse osmosis,microfiltration, ultrafiltration, nanofiltration, cake filtrationprocesses (e.g. by means of automatic pressure filters (membrane, frameor chamber) filter presses, (agitated) pressure filters, suctionfilters, (vacuum) belt filters, (vacuum) drum filters, rotary filters,candle filters), centrifugation processes by means of continuously orbatchwise operated centrifuges or filter centrifuges (e.g. invertingfilter centrifuges, scraper centrifuges, pusher-type centrifuges,worm/screen centrifuges, slide centrifuges, separators or decantercentrifuges), processes utilizing gravity, such as flotation,sedimentation, sink-float purification and clarifying. The biomass isremoved from the culture broth preferably by centrifugation by means ofa decanter or by filtration by means of a membrane filtration unit. Theculture broth is subsequently dried. Again, it is possible to employherein any processes and apparatus known to the skilled worker.Particularly suitable are apparatus for thermal drying such asconvection, contact and radiation drying, for example tray, chamber,channel, flat web, plate, rotary drum, free-fall shaft, sieve belt,stream, fluidized bed, paddle, spherical bed, hotplate, thin film, can,belt, sieve drum, screw, tumble, contact disc, infrared, microwave andfreeze driers, spray driers or spray driers with integrated fluidizedbed, which are, if appropriate, heated by means of steam, oil, gas orelectric current and, if appropriate, operated under reduced pressure.Depending on the apparatus, the mode of operation may be continuous orbatchwise. Additionally or in combination therewith, the mechanicalprocesses of solid/liquid separation already indicated above may beused.

However, granulation by extrusion, as disclosed by WO 97/36996 A2, isnot necessary. The drying process renders the foodstuff stable andstorable.

The culture broth is in particular spray-dried. Preference is given tousing for the drying process spray drying as disclosed in DE 101 04 494A1, DE-A-12 11 911 or EP 0 410 236 A1. In addition, reference is made tocf. Römpp Lexikon Chemie CD-ROM Version 2.0, Georg Thieme Verlag, 1999,“Sprühtrocknung” und Römpp Lexikon Biotechnologie, Georg Thieme Verlag,1992, “Zerstäubungstrocknung” Spray drying has the advantage of a shortdwell time of the product in the hot zone of the drier, thus achieving aparticularly gentle drying process.

Intake temperatures of approx. 115° C.-180° C., preferably 120° C.-130°C., and exhaust temperatures of approx. 50° C.-80° C., preferably 55°C.-70° C., are chosen for spray drying. The preferred gas employed inthe drying process is nitrogen.

It is possible, if appropriate, to add flow aids such as silicic acidsetc., to achieve better flowability. The use of inert carrier materials,i.e. low molecular-weight inorganic carriers such as NaCl, CaCO₃, Na2SO4or MgSO4, organic carriers such as glucose, fructose, sucrose, dextrinsor starch products (rye, barley, oat flour, wheat semolina bran) isconceivable.

The dried product has a residual moisture of preferably less than 10%,preferably less than 5%, based on the dry weight. Its carotenoid contentis between 0.05 and 20%, in particular 1 and 10%, based on the dryweight.

The foodstuff produced in this way may either be used directly or beprocessed by means of further additives, as is likewise disclosed in DE101 04 494 A1.

According to the alternative IIC, the biomass, after it has beencultured and before it is being dried, is first removed from the culturebroth. To this end, any solid/liquid separation methods familiar to andusually employable by the skilled worker, as already mentioned above fordehydration, may be employed. The biomass is removed from the culturebroth preferably by centrifugation by means of a decanter or by membranefiltration.

Subsequently, the biomass is optionally washed with a solvent in whichcarotenoids are not soluble, in particular water, whereby in particularwater-soluble components are removed. This step may, if appropriate, besupplemented using further solvents in which carotenoids are not soluble(e.g. alcohols), although this is not necessary within the scope of theinvention and is not preferred, in order to avoid waste.

Subsequently, sterilization and subsequent or concomitant disruption ofthe cells in the biomass are carried out. Sterilization kills themicroorganisms and stops enzyme activity which may be present. This isimportant for stability and for avoiding degradation of the biomass orthe substances present therein, in particular the carotenoids.

Sterilization may be carried out using a customary method familiar tothe skilled worker. This includes sterilization using steam, inparticular at temperatures of more than 120° C. under pressure (≧1 bar)and for ≧approx. 20 min, and also treatment with high-energy radiationsuch as UV, microwave, gamma or beta rays. The sterilization within theframework of the method of the invention is preferably carried out usingsteam or microwave radiation.

The subsequent or concomitant cell disruption releases the carotenoidspresent in the cells. Cell disruption may likewise be carried out usingany customary processes known to the skilled worker. These includemechanical and nonmechanical methods. The mechanical methods include drymilling, wet milling, stirring, homogenizing (e.g. in a high pressurehomogenizer) and the use of ultrasound or microwaves. Suitablenonmechanical methods are physical, chemical and biochemical methods.These include short time heating, short time freezing, osmotic shock,drying, treatment with acids or bases and enzymic disruption.Advantageously, however, the process used for sterilization is used forcell disruption. Thus, preference is likewise given to carrying out celldisruption using steam or microwave radiation.

Sterilization and/or cell disruption may be carried out continuously orbatchwise.

Sterilization and/or cell disruption may be carried out in thebioreactor used for cultivation or in other apparatus such as autoclavesetc. If the procedure is continuous, it is possible to use themicrowave-using process disclosed in WO 01/83437 A1 and correspondingapparatus.

Prior to extraction, the biomass is, if appropriate, dried and/orhomogenized. Again, it is possible to employ herein any customaryprocesses and devices known to the skilled worker. Particularly suitableare apparatus for thermal drying such as convection, contact andradiation drying, for example tray, chamber, channel, flat web, plate,rotary drum, free-fall shaft, sieve belt, stream, fluidized bed, paddle,spherical bed, hotplate, thin film, can, belt, sieve drum, screw,tumble, contact disc, infrared, microwave and freeze driers, spraydriers or spray driers with integrated fluidized bed, which are, ifappropriate, heated by means of steam, oil, gas or electric current and,if appropriate, operated under reduced pressure. Depending on theapparatus, the mode of operation may be continuous or batchwise.Additionally or in combination therewith, the mechanical processes ofsolid/liquid separation already indicated above may be used.

However, granulation by extrusion, as disclosed by WO 97/36996 A2, isnot necessary.

Subsequently, the carotenoids are partially extracted from the disruptedbiomass by means of a carotenoid-dissolving solvent and separation ofsaid solvent from the biomass. Both the solvent and the biomass thencomprise carotenoids, the majority of said carotenoids being preferablypresent in the solvent.

The highly pure carotenoids are then isolated from the solvent, whereasthe biomass is further processed to give a high quality,carotenoid-containing foodstuff which, due to the preceding celldisruption, also has good carotenoid bioavailability.

Accordingly, partial extraction means the deliberately incompleteextraction of the carotenoids from the biomass (cf. above). Preferenceis thus given to less than 100% of the total amount of carotenoids inthe biomass being extracted from the latter by said extraction withinthe scope of the invention.

This is of great advantage, since complexity of the extraction increasesdisproportionately with the decreasing amount of carotenoid in thebiomass.

The solvents used for extraction are ones which dissolve carotenoidssuch as, for example, hexane, ethyl acetate, dichloromethane orsupercritical carbon dioxide. The preferred solvent used according tothe invention is dichloromethane or supercritical carbon dioxide, itbeing possible, when using supercritical carbon dioxide, to subsequentlytransfer the carotenoids present therein to dichloromethane or to obtainthe product of interest directly by expanding the carbon dioxide. Inthis connection, the amounts of solvents and the mixing times are chosenso that the desired amount of carotenoids is extracted from the biomass.More specifically, the extraction step is carried out only once, thisbeing technically and economically sensible (cf. above).

The extraction may be carried out using any customary processes andapparatus. More specifically, liquid/liquid extraction is carried out ifthe biomass has been disrupted but not dried (carotenoid is present inliquid cell components in soluble form and is extracted therefrom), andsolid/liquid extraction is carried out if the biomass has been dried. Itis possible to use cold and hot extraction within particular temperatureranges, both continuous (e.g. Soxhlet extraction, perforation andpercolation) and discontinuous processes which include, for example,extracting by shaking, with bases, by boiling, and digestion. They mayalso be carried out in a counterflow process.

It is possible to use for liquid/liquid extraction, for example, bubblecolumns, pulsating columns, columns with rotating internal fittings,mixer-settler batteries or stirred tanks etc.

Solid/liquid extraction may be carried out by means of customaryapparatus. Preference is given to using stirred tanks or mixer-settlerapparatus.

Alternatively, the cells may be disrupted without prior removal of thefermentation medium, followed by direct separation of a resultantcarotenoid suspension from the biomass, for example by means of adecanter. The carotenoid suspension is subsequently taken up indichloromethane and processed further or, alternatively, purified bywashing with various aqueous solutions.

The highly pure carotenoids are isolated from the solvent bycrystallizing said carotenoids from the solvent used and isolating thecarotenoid crystals, in particular by filtration. The remaining motherliquor may, after distillation, reenter into the process, thusminimizing product losses despite low effort.

Crystallization may be carried out as usual. In addition, reference ismade to cf. Römpp Lexikon Chemie CD-ROM Version 2.0, Georg ThiemeVerlag, 1999, “Kristallisation”.

Crystallization is preferably carried out by gradually replacing thesolvent with a solvent in which carotenoids are not soluble. Thus,carotenoid solubility is continuously decreased until said carotenoidsprecipitate in the form of pure crystals. Preference is given here tousing a “lower alcohol” or water. Lower alcohol means aliphatic alcoholshaving from 1 to 4 carbon atoms. These include methanol, ethanol,propanol, isopropanol, 1-butanol, tert-butanol and sec-butanol.Preference is given to using methanol.

In this connection, the carotenoid solution may be heated, thetemperature being kept preferably <100° C., in particular <60° C., sothat dichloromethane is distilled off. It is also conceivable to usereduced pressure. The carotenoid crystals are then isolated and this maybe carried out by the usual measures, in particular by filtration. Ifdesired, further optional drying and/or purification steps may follow.These are, however, not necessary, since the carotenoid crystals arealready highly pure.

The carotenoids are obtained as highly pure crystals and have a purityof at least 95%, preferably >95%, preferentially >96%, particularlypreferably >97%, very particularly preferably >98%, most preferably>99%.

The achievable yields are between 45% and 95%, preferably between 70%and 95%, based on the amount present in the culture broth (0.5-15 g/L,preferably 1-10 g/L).

In order to further process the likewise carotenoid-containing biomassto give a high quality foodstuff, first residual solvents are removedfrom the carotenoid-containing biomass. This is preferably carried outby way of steam distillations or “stripping” with steam (cf. RömppLexikon Chemie CD-ROM Version 2.0, Georg Thieme Verlag, 1999,“Strippen”).

This may be followed, if appropriate, by homogeneously suspending thebiomass in the culture broth removed above, in which case a solidcontent of >100 g/L and <600 g/L should be observed in order for thesubsequent drying of the biomass or suspension for producing thefoodstuff to be carried out without technical difficulties, i.e. thesuspension must be pumpable. Suitable drying processes are all of theprocesses and apparatus already mentioned. More specifically, spraydrying is used for the drying process which may be carried out asdisclosed in DE 101 04 494 A1.

Intake temperatures of approx. 100° C.-180° C., preferably 120° C.-130°C., and exhaust temperatures of approx. 50° C.-80° C., preferably 55°C.-70° C., are chosen for spray drying. The preferred gas employed inthe drying process is nitrogen.

The foodstuff produced in this way may either be used directly or beprocessed by means of further additives, as is likewise disclosed in DE101 04 494 A1.

Foodstuffs are regarded as compositions used for nutrition. These alsoinclude compositions for supplementing nutrition. Animal feedstuffs andanimal feed supplements, in particular, are regarded as foodstuffs. Inaddition, reference is made to Römpp Lexikon Chemie CD-ROM Version 2.0,Georg Thieme Verlag, 1999, “Nahrungsmittel”.

The dry product has a residual moisture of preferably less than 5%,based on dry weight. Its carotenoid content is between 0.05 and 20%, inparticular 1 and 10%, based on dry weight. The desired carotenoidcontent can be controlled via the degree of extraction (cf. above).

Thus, foodstuffs obtainable by the method according to the inventioninclude already after production large amounts of carotenoids which neednot be added. Moreover, the nutrient content of said foodstuff isincreased, due to the fact that it also contains biomass, in addition tothe at least one carotenoid. The increase in nutrient content isparticularly large according to the preferred alternative, since itincludes, aside from the at least one carotenoid and biomass, inaddition all media components of the fermentation. As a result, thereare virtually no waste products, apart from aqueous media, which,however, can be purified without problems in a purification plant. Inaddition, the entire amount of carotenoids produced without or with onlymarginal losses is used, since no separation or work-up steps with heavylosses need to be carried out, in order to extract the total amount ofcarotenoid.

All of the solvents used in the above-described method of the inventionare purified as far as possible and subsequently reused or reenteredinto the process. More specifically, the dichloromethane used ispurified already during solvent replacement and is thereafter ready tobe used again. The lower alcohol or methanol is purified, for example,by distillation and is likewise reused. The only waste produced is thedistillation bottom which, together with the aqueous media, may bedirected without risk to a purification plant where the actual wasteproduced in the end is only a small amount of sludge. Thus, the method

described is essentially waste-free. The invention is illustrated inmore detail below on the basis of examples.

A) Cultivation of Blakeslea trispora

The following media were used for fermentation of Blakeslea trispora toproduce the carotenoids: Medium 1: Glucose 10.00 g/l Cotton seed oil30.00 g/l Soybean oil 30.00 g/l Dextrin 60.00 g/l Cottonseed meal 75.00g/l Triton X 100 1.20 g/l Ascorbic acid 6.00 g/l Lactic acid 2.00 g/lKH₂PO₄ 0.50 g/l MnSO₄ × H2O 100 mg/l Thiamine-HCl 2 mg/l Isoniazide(isonicotinic acid hydrazide) 0.75 g/lThe pH was adjusted to 6.5.

Medium 2: Glucose 20 g/l Asparagine 2.00 g/l KH₂PO₄ 5.00 g/l MgSO₄ ×7H₂O 0.50 g/l CaCl₂ 28 mg/l Thiamine-HCl 1.00 mg/l Citric acid 2.00 mg/lFe(NO₃)₃ × 9H₂O 1.50 mg/l ZnSO₄ × 7H₂O 1.00 mg/l MnSO₄ × H₂O 0.30 mg/lCuSO₄ × 5H₂O 0.05 mg/l Na₂MoO₄ × 2H₂O 0.05 mg/l

Medium 3 Glucose 70.00 g/l Asparagine 2.00 g/l Yeast extract 1.00 g/lKH₂PO₄ 1.50 g/l MgSO₄ × 7H₂O 0.50 g/l Span 20 1.00 g/l Thiamine-HCl 5.0mg/lThe pH was adjusted to 5.5.

200 ml of the media described were inoculated in each case with sporesuspensions of Blakeslea trispora ATCC 14272 Mating Type (−), whichcomprised 10⁸ (for Medium 2) and, respectively, 10⁷ (for Medium 1 and 3)spores. The cultivation was carried out in each case in 1 l Erlenmeyerflasks with baffles. With each medium, six identical flasks wereprepared and incubated on a shaker at 28° C. and 140 rpm for 7 days.

B) Genetic Modification of Blakeslea Trispora

Materials and Methods

Molecular genetics work was carried out, unless described otherwise, bythe methods in Current Protocols in Molecular Biology (Ausubel et al.,1999, John Wiley & Sons).

Strains and Growth Conditions

The Blakeslea trispora strains ATCC 14271 (mating type (+)) andATCC14272 (−) (a wild type) were obtained, mating type (−)) wereobtained from the American Type Culture Collection. B. trispora weregrown in MEP medium (malt extract-peptone medium): 30 g/l malt extract(Difco), 3 g/l peptone (Soytone, Difco), 20 g/l agar, pH set to 5.5, ad1000 ml with H₂O at 28° C.

Agrobacterium tumefaciens LBA4404 were grown according to Hoekema et al.(1983, Nature 303:179-180) at 28° C. for 24 h in agrobacterial minimalmedium (AMM): 10 mM K₂HPO₄, 10 mM KH₂PO₄, 10 mM glucose, MM salts (2.5mM NaCl, 2 mM MgSO₄, 700 μM CaCl₂, 9 μM FeSO₄, 4 mM (NH₄)₂SO₄).

Transformation of Agrobacterium tumefaciens

The plasmid pBinAHyg was electroporated into the agrobacterial strainLBA 4404 (Hoekema et al., 1983, Nature 303:179-180) (Mozo and Hooykaas,1991, Plant Mol. Biol. 16:917-918). The following antibiotics were usedfor selection during agrobacterial growth: Rifampicin 50 mg/l (selectionfor the A. tumefaciens chromosome), streptomycin 30 mg/l (selection forthe helper plasmid) and kanamycin 100 mg/l (selection for the binaryvector).

Transformation of Blakeslea trispora

After 24 h of growth in AMM, the agrobacteria were diluted fortransformation to an OD₆₀₀ of 0.15 in induction medium (IM: MM salts, 40mM MES (pH 5.6), 5 mM glucose, 2 mM phosphate, 0.5% glycerol, 200 μMacetosyringone) and grown again in IM to an OD₆₀₀ of approx. 0.6overnight.

For coincubation of Blakeslea ATCC 14271 or ATCC14272 and Agrobacterium,100 μl of agrobacterial suspension were mixed with 100 μl of Blakesleaspore suspension (10⁷ spores/ml in 0.9% NaCl) and distributed in asterile manner on a nylon membrane (Hybond N, Amersham) on IM-agaroseplates (IM+18 g/l agar). After 3 days of incubation at 26° C., themembrane was transferred to an MEP-agar plate (30 g/l malt extract, 3g/l peptone, pH 5.5, 18 g/l agar). To select for transformed Blakesleacells, the medium comprised hygromycin at a concentration of 100 mg/land, to select against agrobacteria, 100 mg/l cefotaxime. The incubationwas carried out at 26° C. for approx. 7 days. This was followed bytransferring mycelium to fresh selection plates. Resultant spores wererinsed with 0.9% NaCl and plated on CM17-1 agar (3 g/l glucose, 200 mg/lL-asparagine, 50 mg/l MgSO₄×7H₂O, 150 mg/l KH₂PO₄, 25 μg/l thiamine-HCl,100 mg/l Yeast Extract, 100 mg/l sodium deoxycholate, 100 mg/Lhygromycin, 100 mg/L cefotaxime, pH 5.5, 18 g/l agar). Individualgenetically modified spores were isolated by putting them individuallyon selection medium, using an FACS instrument from BectonDickson (ModellVantage+Diva Option).

Mutagenesis with MNNG

To reduce the number of nuclei per spore, spore suspensions were treatedwith MNNG (N-methyl-N′-nitro-N-nitrosoguanidine). For this, first aspore suspension containing 1×10⁷ spores/ml in Tris/HCl buffer, pH 7.0was prepared. The spore suspension was admixed with MNNG at a finalconcentration of 100 μg/ml. The time of incubation in MNNG was chosen insuch a way that the survival rate of the spores was approx. 5%. Afterincubation with MNNG, the spores were washed three times with 1 g/l Span20 in 50 mM phosphate buffer pH 7.0 and plated.

Selection of Homonuclear Cells

Homonuclear Blakeslea trispora carB cells were selected in a mannersimilar to the experimental protocol for Phycomyces blakesleeanus(Roncero et al., 1984, Mutation Research, 125:195-204), modified bygrowth in the presence of 5-carbon-5-deazariboflavin (1 μg/ml) andHygromycin 100 (μg/ml).

Preparation of Genetically Modified Blakeslea trispora byagrobacterium-Mediated Transformation

Preparation of the Recombinant Plasmid pBinAHyg

The gpdA-hph-trpC-cassette was isolated as BglII/HindIII fragment fromthe plasmid pANsCos1 (FIG. 1, Osiewacz, 1994, Curr. Genet. 26:87-90, SEQID NO: 4) and ligated into the binary plasmid pBin19 (Bevan, 1984,Nucleic Acids Res. 12:8711-8721) opened with BamHI/HindIII. The vectorobtained in this way was referred to as pBinAHyg (FIG. 2, SEQ ID NO: 3)and comprised the E. coli hygromycin resistance gene (hph) under thecontrol of the gpd promoter (SEQ ID NO: 1) and the trpC terminator (SEQID NO: 2) from Aspergillus nidulans and the corresponding bordersequences required for Agrobacterium DNA transfer. The vectors mentionedin the exemplary embodiments described hereinbelow are pBinAHygderivatives.

Transfer of pBinAHyg and pBinAHyg Derivatives into Agrobacteriumtumefaciens

The transfer of the pBinAHyg plasmid into agrobacteria is described byway of example below. The derivatives were transferred in a similarmanner.

The plasmid pBinAHyg was electroporated into the agrobacterial strainLBA 4404 (Hoekema et al., 1983, Nature 303:179-180) (Mozo and Hooykaas,1991, Plant Mol. Biol. 16:917-918). The following antibiotics were usedfor selection during agrobacterial growth: Rifampicin 50 mg/l (selectionfor the A. tumefaciens chromosome), streptomycin 30 mg/l (selection forthe helper plasmid) and kanamycin 100 mg/l (selection for the binaryvector).

Transfer of pBinAHyg and pBinAHyg Derivatives into Blakeslea trispora

After 24 h of growth in AMM, the agrobacteria were diluted fortransformation to an OD₆₆₀ of 0.15 in induction medium (IM: MM salts, 40mM MES (pH 5.6), 5 mM glucose, 2 mM phosphate, 0.5% glycerol, 200 μMacetosyringone) and grown again in IM to an OD₆₆₀ of approx. 0.6overnight.

For coincubation of Blakeslea trispora (B.t.) and Agrobacteriumtumefasciens (A.t.) 100 μl of agrobacterial suspension were mixed with100 μl of Blakeslea spore suspension (10⁷ spores/ml in 0.9% NaCl) anddistributed in a sterile manner on a nylon membrane (Hybond N, Amersham)on IM-agarose plates (IM+18 g/l agar). After 3 days of incubation at 26°C., the membrane was transferred to an MEP-agar plate (30 g/l maltextract, 3 g/l peptone, pH 5.5, 18 g/l agar).

To select for transformed Blakeslea cells, the medium containedhygromycin at a concentration of 100 mg/l and, to select againstagrobacteria, 100 mg/l cefotaxime. The incubation was carried out at 26°C. for approx. 7 days. This was followed by transferring mycelium tofresh selection plates. Resultant spores were rinsed with 0.9% NaCl andplated on CM17-1 agar (3 g/l glucose, 200 mg/l L-asparagine, 50 mg/lMgSO₄×7H₂O, 150 mg/l KH₂PO₄, 25 μg/l thiamine-HCl, 100 mg/l YeastExtract, 100 mg/l sodium deoxycholate, pH 5.5, 100 mg/L cefotaxime, 100mg/L hygromycine, 18 g/l agar). The transfer of spores to freshselection plates was repeated three times. In this way, the transformantBlakeslea trispora GMO 3005 was isolated. Alternatively, the GMO(genetically modified organisms) were selected by applying the sporesindividually to CM-17 agar containing 100 mg/l cefotaxime, 100 mg/lhygromycin, by means of the BectonDickinson FacsVantage+Diva Option. Inthis case, fungal mycelium formed only where the spores had beengenetically modified.

Detection of the Genetic Modification Due to Transfer of pBinAHyg andpBinAHyg Derivatives in Blakeslea trispora

Detection of the transfer is described by way of example below forpBinAHyg in Blakeslea trispora. Detection of the transfer of thederivatives was carried out in a similar manner.

200 ml of MEP medium (30 g/l malt extract, 3 g/l peptone, pH 5.5) wereinoculated with 10⁵ to 10⁷ spores of the Blakeslea trispora GMO 3005transformant and incubated on a rotary shaker at 200 rpm and 26° C. for7 days. To detect successful transformation, DNA was isolated from themycelium (Peqlab Fungal DNA Mini Kit) and used in a PCR (program: 94° C.for 1 min, then 30 cycles of 1 min. at 94° C., 1 min. at 58° C., 1 min.at 72° C., each).

The primers hph-forward (5′-CGATGTAGGAGGGCGTGGATA, SEQ ID NO: 5) andhph-reverse (5′-GCTTCTGCGGGCGATTTGTGT, SEQ ID NO: 6) were used fordetecting the hygromycin resistance gene (hph). The expected hphfragment was 800 bp in length.

The primers nptIII-forward (5′-TGAGAATATCACCGGAATTG, SEQ ID NO: 7) andnptIII-reverse (5′-AGCTCGACATACTGTTCTTCC, SEQ ID NO: 8) were used foramplification of the kanamycin resistance gene nptIII and thus as acontrol for agrobacteria. The expected nptIII fragment was 700 bp inlength.

The primers MAT292 (5′-GTGAATGGAAATCCCATCGCTGTC, SEQ ID NO: 9) andMAT293 (5′-AGTGGGTACTCTAAAGGCCATACC, SEQ ID NO: 10) were used foramplification of a fragment of the glycerinaldehyde 3-phosphatedehydrogenase gene gpd1 and thus as a control for Blakeslea trispora.The expected gpd1 fragment was 500 bp in length.

FIG. 3 depicts the result of the PCR of Blakeslea trispora DNA on thebasis of a standard gel. The gel lanes were loaded as follows: 1) 100 bpsize marker(100 bp-1 kb) 2) B.t. GMO 3005 primer nptIII-for/nptIII-rev3) B.t. GMO 3005 primer hph-for/hph-rev 4) B.t. GMO 3005 primerMAT292/MAT293 (gpd) 5) A.t. with pBinAHyg plasmid primernptIII-for/nptIII-rev 6) A.t. with pBinAHyg plasmid primerhph-for/hph-rev 7) B.t. 14272 WT primer nptIII-for/nptIII-rev 8) B.t.14272 WT primer hph-for/hph-rev 9) B.t. 14272 WT primer MAT292/MAT293(gpd)

The hygromycin resistance gene (hph) and, as a positive control, theglycerinaldehyde 3-phosphate dehydrogenase gene (gpd1) were detected inBlakeslea trispora DNA. In contrast, nptIII was not detected.

Thus, the genetic modification of Blakeslea trispora byAgrobacterium-mediated transformation was detected.

Isolation of Homokaryotic Blakeslea trispora GMOs:Preparation ofHomonuclear Strains

The successful transfer of the pBinAHyg vector and pBinAHyg derivativesinto Blakeslea trispora produces genetically modified organisms. In GMOof Blakeslea trispora. However, Blakeslea has multinuclear cells at allstages of the vegetative and sexual cell cycle. Therefore, vectorforeign DNA is usually inserted only in one nucleus. It is, however, theaim to obtain Blakeslea strains in which vector foreign DNA has beeninserted in all nuclei, i.e. the aim is a homonuclear recombinant fungalmycelium.

In order to prepare homokaryotic cells of this kind, spore suspensionsof the recombinant strains were first treated with MNNG. For this, firsta spore suspension containing 1×10⁷ spores/ml in Tris/HCl buffer, pH 7.0was prepared. The spore suspension was admixed with MNNG at a finalconcentration of 100 μg/ml. The time of incubation in MNNG was chosen insuch a way that the survival rate of the spores was approx. 5%. Afterincubation with MNNG, the spores were washed three times with 1 g/l Span20 in 50 mM phosphate buffer pH 7.0 and plated.

1) Preparation of Homonuclear Recombinant Strains by Means of FACS(Fluorescence-Activated Cell Sorting)

A small proportion of the spores of Blakeslea trispora or of thegenetically modified Blakeslea trispora strains is by naturemononuclear. To produce homonuclear recombinant strains comprising theforeign DNA of pBinAHyg or pBinAHyg derivatives, the mononuclear sporeswere sorted out by means of FACS and plated on MEP (30 g/l malt extract,3 g/l peptone, pH 5.5, 18 g/l agar) containing 100 mg/l cefotaxime and100 mg/l hygromycin. The mycelia produced here were homonuclear. ForFACS, the spores of a 3 day old smear were washed off with 10 ml ofTris-HCl 50 mMol+0.1% Span20 per agar plate. The spore concentration wasfrom 0.5 to 0.8×10⁷ spores per ml. 1 ml of DMSO and 10 μl of Syto 11(dye stock solution in DMSO, Molecular Probes No. S-7573) were added to9 ml of spore suspension. This was followed by staining at 30° C. for 2h. Selection and application were carried out by means of aBectonDickinson FacsVantage+Diva Option. First, a size selection wascarried out in order to separate individual spores from aggregates andcontaminations. These spores were then applied sorted according to theirfluorescence (excitation=488 nm emission=530 nm). The left shoulder ofthe Gauss curve of the fluorescence frequency distribution contained themononuclear spores.

The spores were then plated on MEP agar plates and new spores weregenerated.

These spores were plated on medium comprising 5-carbon-5-deazariboflavinand, additionally, hygromycin, in a similar manner to the protocol byRoncero et al.

This enabled homokaryotic cells of the genotype hyg^(R) and dar⁻ to beselected.

2) Preparation of Homonuclear Strains by Reducing the Number of Nucleiand Selection with FACS

To reduce the number of nuclei per spore, spore suspensions were treatedwith MNNG (N-methyl-N′-nitro-N-nitrosoguanidine) prior to selection,thus achieving a reduction in the number of nuclei by chemicalmutagenesis.

For this, first a spore suspension containing 1×10⁷ spores/ml inTris/HCl buffer, pH 7.0 was prepared. The spore suspension was admixedwith MNNG at a final concentration of 100 μg/ml. The time of incubationin MNNG was chosen in such a way that the survival rate of the sporeswas approx. 5%. After incubation with MNNG, the spores were washed threetimes with 1 g/l Span 20 in 50 mM phosphate buffer pH 7.0 and sorted andselected by the method described under 1).

As an alternative, it was also possible to reduce the number of nucleiin the spores by using X-rays and UV rays, as described by Cerdá-Olmedoand Patricia Reau in Mutation Res., 9(1970), 369-384.

3) Preparation of Homonuclear Strains by Selection for RecessiveSelection Markers

A suitable recessive selection marker for selection of homonuclearmycelia is, for example, the recessive selection marker pyrG. Wild-typestrains of Blakeslea trispora are pyrG⁺. These strains are unable togrow in the presence of the pyrimidine analog 5-fluoroorotate (FOA),because they convert FOA to lethal metabolites via orotidine5′-monophosphate decarboxylase. Genetically modified pyrG⁻ homonuclearBlakeslea lack the enzyme activity of orotidine 5′-monophosphatedecarboxylase. Consequently, these pyrG⁻ strains are unable to utilize5-fluoroorotate. Therefore, these strains grow in the presence of FOAand uracil. If the pyrG⁻ mutation and the foreign DNA insert are coupledon the nucleus of a mononuclear spore, this spore may form homonuclearrecombinant fungal mycelium.

First, the plasmid pBinAHygBTpyrG-SCO (SEQ ID NO: 36, FIG. 4) wasgenerated by inserting a fragment of pyrG (SEQ ID NO: 65) from Blakesleatrispora into pBinAHyg. Said plasmid was transformed into Blakesleatrispora and caused pyrG disruption there due to homologousrecombination.

Homonuclear Blakeslea trispora GMO with the pyrG phenotype were selectedas follows. Plating on MEP (30 g/l malt extract, 3 g/l peptone, pH 5.5,18 g/l agar) containing 100 mg/l cefotaxime and 100 mg/l hygromycin foragrobacterium-mediated transformation of pBinAHygBTpyrG-SCO was carriedout as described above. The spores of the transformants were washed offwith 10 ml of Tris-HCl 50 mM+0.1% Span20 per agar plate. The sporeconcentration was from 0.5 to 0.8×10⁷ spores per ml. The spores werethen plated on FOA medium containing 100 mg/l cefotaxime and 100 mg/lhygromycin. FOA medium comprised, per liter, 20 g of glucose, 1 g ofFOA, 50 mg of uracil, 200 ml of citrate buffer (0.5 M, pH 4.5) and 40 mlof trace salt solution according to Sutter, 1975, PNAS, 72:127).Homonuclear pyrG⁻ mutants exhibited growth on the uracil-containing FOAmedium but no growth when plated on FOA medium without uracil. In thesame way, homonuclear GMO were prepared from the Blakeslea trispora GMOdescribed below for producing xanthophylls.

Alternatively, it is possible to plate the spores according to theprotocol by Roncero et al. on medium comprising5-carbon-5-deazariboflavin and, additionally, hygromycin (Roncero etal., 1984, Mutation Research, 125: 195-204). This enables homokaryoticcells of the genotype hyg^(R) and dar⁻ to be selected.

According to this principle, homokaryotic Blakeslea trispora strainswith the phenotype hyg^(R) and dar⁻ are generated.

Exemplary Embodiments for Preparing Genetically Modified Organisms ofBlakeslea trispora for Producing Carotenoids and Carotenoid Precursors.

The plasmids mentioned below were generated by the “overlap-extensionPCR” method and by subsequent insertion of the amplification productsinto the pBinAHyg plasmid. The overlap-extension PCR method was carriedout as described in Innis et al. (Eds) PCR protocols: a guide to methodsand applications, Academic Press, San Diego. Transformation of thepBinAHyg derivatives and preparation of homonuclear genetically modifiedBlakeslea trispora strains were carried out as described above.

Genetically Modified Blakeslea trispora Strains for Producing Zeaxanthin

The following plasmids (pBinAHyg derivatives) were used for geneticmodification of Blakeslea trispora for the production of zeaxanthin, andthus encode inter alia hydroxylases (crtZ):

-   -   ptef1-HPcrtZ, comprising the gene of the HPcrtZ hydroxylase (SEQ        ID NO:70) from Haematococcus pluvialis Flotow NIES-144        (Accession No. AF162276) under the control of the Blakeslea        trispora ptef1 promoter (Seq. pBinAHygBTpTEF1-HPcrtZ, SEQ ID        NO:37, FIG. 5);    -   p-carRA-HPcrtZ, comprising the gene of the HPcrtZ hydroxylase        from Haematococcus pluvialis Flotow NIES-144 under the control        of the Blakeslea trispora pcarRA promoter (Seq.        pBinAHygBTpcarRA-HPcrtZ, SEQ ID NO:38, FIG. 6);    -   p-carB-HPcrtZ, comprising the gene of the HPcrtZ hydroxylase        from Haematococcus pluvialis Flotow NIES-144 under the control        of the Blakeslea trispora pcarB promoter (Seq.        pBinAHygBTpcarB-HPcrtZ, SEQ ID NO:39, FIG. 7);    -   p-carRA-HPcrtZ-TAG-3′carA-IR, comprising the gene of the HPcrtZ        hydroxylase from Haematococcus pluvialis Flotow NIES-144 under        the control of the Blakeslea trispora pcarRA promoter. An        inverted repeat structure is located downstream of the        hydroxylase gene, which structure is derived from the 3′ end of        carA and the region downstream of carA (IR, SEQ ID NO:74,        “Inverted Repeat 1” approx. 350 bp of carA, then approx. 200 bp        “Loop” and then approx. 350 bp “Inverted Repeat 2”) (Seq.        pBinAHyg-BTpcarRA-HPcrtZ-TAG-3′carA-IR, SEQ ID NO:40, FIG. 8);    -   p-carRA-HPcrtZ-GCG-3′carA-IR, comprising the gene of the HPcrtZ        hydroxylase from Haematococcus pluvialis Flotow NIES-144 under        the control of the Blakeslea trispora pcarRA promoter. The        hydroxylase gene is fused to an inverted repeat structure which        is derived from the 3′ end of carRA and the region downstream of        carA (IR, SEQ ID NO:74, “Inverted Repeat 1” approx. 350 bp of        carA, then approx. 200 bp “Loop” and then approx. 350 bp        “Inverted Repeat 2”). Consequently, the derived fusion protein        consists of the Haematococcus pluvialis hydroxylase and the        carboxy terminus of Blakeslea trispora CarA (Seq.        pBinAHyg-BTpcarRA-HPcrtZ-GCG-3′carA-IR, SEQ ID NO:41, FIG. 9).    -   p-tef1-EUcrtZ, comprising the gene of the EUcrtZ hydroxylase        (SEQ ID NO:71) from Erwinia uredova 20D3 (Accession No. D90087)        under the control of the ptef1 promoter (Seq.        pBinAHygBTpTEF1-EUcrtZ, SEQ ID NO:42, FIG. 10);    -   p-carRA-EUcrtZ, comprising the gene of the EUcrtZ hydroxylase        from Erwinia uredova 20D3 under the control of the Blakeslea        trispora pcarRA promoter (Seq. pBinAHygBTpcarRA-EUcrtZ, SEQ ID        NO:43, FIG. 11);    -   p-carB-EUcrtZ, comprising the gene of the EUcrtZ hydroxylase        from Erwinia uredova 20D3 under the control of the Blakeslea        trispora pcarB promoter (Seq. pBinAHygBTpcarB-EUcrtZ, SEQ ID        NO:44, FIG. 12);    -   p-gpdA-HPcrtZ-t-crtZ, comprising the gene of the HPcrtZ        hydroxylase from Haematococcus pluvialis Flotow NIES-144 under        the control of the gpdA promoter and the t-crtZ terminator; i.e.        of the sequence section downstream of crtZ from Haematococcus        pluvialis Flotow NIES-144 (SEQ ID NO:73) (Seq.        pBinAHyg-gpdA-HPcrtZ-tcrtZ, SEQ ID NO:43, FIG. 13).    -   p-gpdA-BTcarR-HPcrtZ-BTcarA, comprising a gene fusion of genes        of lycopine cyclase carR from Blakeslea trispora, of HPcrtZ        hydroxylase from Haematococcus pluvialis Flotow NIES-144 and of        the phytoene synthase carA from Blakeslea trispora and under the        control of the Aspergillus nidulans gpdA promoter (Seq.        pBinAHyg-carR_crtZ_carA, SEQ ID NO:46, FIG. 14).        Preparation of Genetically Modified Blakeslea trispora Strains        for Producing Canthaxanthin

The following plasmids (pBinAHyg derivatives) were used for geneticmodification of Blakeslea trispora for the production of canthaxanthin,and thus encode inter alia ketolases (crtW):

-   -   p-tef1-NPcrtW, comprising the gene of the NPcrtW ketolase (SEQ        ID NO:72) from Nostoc punctiforme PCC73102 (ORF148, Accession        No. NZ_AABC01000196) and under the control of the Blakeslea        trispora ptef1 promoter (Seq. pBinAHygBTpTEF1-NpucrtW, SEQ ID        NO:47, FIG. 15);    -   p-carRA-NPcrtW, comprising the gene of the NPcrtW ketolase from        Nostoc punctiforme PCC73102 and under the control of the        Blakeslea trispora pcarRA promoter (Seq.        pBinAHygBTpcarRA-NpucrtW, SEQ ID NO:48, FIG. 16);    -   p-carB-NPcrtW, comprising the gene of the NPcrtW ketolase from        Nostoc punctiforme PCC73102 and under the control of the        Blakeslea trispora pcarB promoter (Seq. pBinAHygBTpcarB-NpucrtW,        SEQ ID NO:49, FIG. 17).        Preparation of Genetically Modified Blakeslea trispora Strains        for Producing Astaxanthin

The following plasmids (pBinAHyg derivatives) were used for geneticmodification of Blakeslea trispora for producing astaxanthin, i.e.encode inter alia hydroxylases (crtZ) and ketolases (crtW):

-   -   p-carRA-HPcrtZ-pcarRA-NPcrtW, comprising the gene of the HPcrtZ        hydroxylase from Haematococcus pluvialis Flotow NIES-144 and the        gene of the NPcrtW ketolase from Nostoc punctiforme PCC73102        (ORF148, Accession No. NZ-AABC01000196), both in each case under        the control of the Blakeslea trispora pcarRA promoter (Seq.        pBinAHygBTpcarRA-HPcrtZ-BTpcarRA-NpucrtW, SEQ ID NO:50, FIG.        18);    -   p-carRA-EUcrtZ-pcarRA-NPcrtW, comprising the gene of the EUcrtZ        hydroxylase from Erwinia uredova 20D3 (Accession No. D90087) and        the gene of the NPcrTW ketolase from Nostoc punctiforme        PCC73102, both in each case under the control of the Blakeslea        trispora pcarRA promoter (Seq.        pBinAHygBTpcarRA-EUcrtZ-BTpcarRA-NpucrtW, SEQ ID NO:51, FIG.        19).        Cloning and Sequence Analysis of Genes and Promoters which May        be Utilized by Way of Example for Genetic Modification of        Blakeslea trispora.

Cloning and sequencing of various Blakeslea trispora genes and promotersare described by way of example below.

Cloning and Sequence Analysis of ptef1

Blakeslea trispora p-tef was cloned on the basis of a sequence,previously published in GenBank, of the structural gene of Blakesleatrispora translation elongation factor 1-α (AF157235). Starting from thesequence entry AF157235 primers were selected for inverted PCR in orderto amplify and sequence the promoter region upstream of said structuralgene. In the inverted nested PCR of 200 ng of XhoI-cleaved andcircularized genomic DNA of Blakeslea trispora ATCC14272, a 3000-bpfragment was obtained in the following reaction mixture: template DNA (1μg of genomic DNA of Blakeslea trispora ATCC 14272) primers MAT3445′-GGCGTACTTGAAGGAACCCTTACCG-3′ (SEQ ID NO: 63) and MAT 3455′-ATTGATGCTCCCGGTCACCGTGATT-3′ (SEQ ID NO: 64), 0.25 μM each, 100 μMdNTP, 10 μl of Herculase polymerase buffer 10×, 5 U of Herculase(addition at 85° C.), H₂O ad 100 μl. The PCR profile was as follows: 95°C., 10 min (1 cycle); 85° C., 5 min (1 cycle); 60° C., 30 s, 72° C., 60s, 95° C., 30 s (30 cycles); 72° C., 10 min (1 cycle). The sequencesection upstream of the putative start codon of the tef1 gene in the3000-bp fragment was referred to as ptef1 promoter.

Cloning, Sequence Analysis of the Gene of HMG-CoA Reductase fromBlakeslea trispora

First, the cosmid vector pANsCos1 was used for preparing a gene libraryof Blakeslea trispora ATCC 14272, Mating Type (−). The vector waslinearized by cleavage with XbaI and then dephosphorylated. Furthercleavage with BamHI generated the insertion site into which theBlakeslea trispora genomic DNA, partially cleaved with Sau3AI anddephosphorylated, was ligated. The cosmids produced in this way weresubsequently packaged in vitro and transferred into Escherichia coli.

On the basis of the known sequence of a fragment of the Blakesleatrispora gene encoding HMG-CoA reductase (Eur. J. Biochem 220, 403-408(1994)), a 315-bp DNA probe was prepared by the following PCR. Reactionmixture: 1 μg of genomic DNA of Blakeslea trispora ATCC 14272, primersMAT314 5′-CCGATGGCGACGACGGAAGGTTGTT-3′ [SEQ ID NO: 79] and MAT3155′-CATGTTCATGCCCATTGCATCACCT-3′ [SEQ ID NO: 80], 0.25 μM each, 100 μMdNTP, 10 μl of Herculase polymerase buffer 10×, 5 U of Herculase(addition at 85° C.), H₂O ad 100 μl. The PCR profile was as follows: 95°C., 10 min (1 cycle); 85° C., 5 min (1 cycle); 58° C., 30 s, 72° C., 30s, 95° C., 30 s (30 cycles); 72° C., 10 min (1 cycle).

This DNA probe was used for screeing the cosmid gene library. A clonewhose cosmid hybridized with said DNA probe was identified. The insertof this cosmid was sequenced. The DNA sequence comprised a section whichwas assigned to the gene of an MHG-CoA reductase [HMG-CoA-Red.gb].

Cloning and Sequence Analysis of carB

(carB=Blakeslea trispora phytoene desaturase gene) The degeneratedprimers MAT182 5′-GCNGARGGNATHTGGTA-3′ (SEQ ID 52) and MAT1925′-TCNGCNAGRAADATRTTRTG-3′ (SEQ ID 53) were derived from comparing thepeptide sequences of phytoene desaturases and comparing thecorresponding DNA sequences of Phycomyces blakesleeanus, Cercosporanicotianae, Phaffia rhodozyma and Neurospora crassa. The PCR was carriedout in 100 μl reaction mixtures. These comprised 200 ng of genomic DNAof Blakeslea trispora ATCC14272, 1 μM MAT182, 1 μM MAT192, 100 μM dNTP,10 μl of Pfu polymerase buffer 10×, 2.5 U of Pfu polymerase (addition at85° C.), H₂O ad 100 μl.

The PCR profile was as follows: 95° C., 10 min (1 cycle); 85° C., 5 min(1 cycle); 40° C., 30 s, 72° C., 30 s, 95° C., 30 s (35 cycles); 72° C.,10 min (1 cycle).

This resulted in a 358-bp fragment whose derived peptide sequence issimilar to the phytoene desaturase sequences. The method of inverted PCR(Innis et al. in PCR protocols: a guide to methods and applications.1990. pp. 219-227) was used for amplifying, cloning and sequencing,according to the principle of chromosome walking, the gene regionsupstream and downstream of the 350-bp fragment as follows:

-   (i) a 1.1 kbp fragment, by PCR with the primers MAT219    5′-AAGTGACACCGGTTACACGCTTGTCTT-3′ (SEQ ID 54) and MAT 220    5′-GCTTATCACCATCTGTTACCTCCTTGC-3′ (SEQ ID 55), obtained from 200 ng    of EcoRI-cleaved and circularized genomic DNA of Blakeslea trispora    ATCC14272, 0.25 μM MAT219, 0.25 μM MAT220, 100 μM dNTP, 10 μl of    Herculase polymerase buffer 10×, 5 U of Herculase (addition at 85°    C.), H₂O ad 100 μl. The PCR profile as as follows: 95° C., 10 min (1    cycle); 85° C., 5 min (1 cycle); 60° C., 30 s, 72° C., 60 s, 95° C.,    30 s (30 cycles); 72° C., 10 min (1 cycle),-   (ii) a 2.9 kbp fragment, by PCR with the primers MAT219 and MAT220,    obtained from 200 ng of XbaI cleaved and circularized genomic DNA    Blakeslea trispora ATCC14272, 0.25 μM MAT219, 0.25 μM MAT220, 100 μM    dNTP, 10 μl of Herculase polymerase buffer 10×, 5 U of Herculase    (addition at 85° C.), H₂O ad 100 μl. The PCR profile was as follows:    95° C., 10 min (1 cycle); 85° C., 5 min (1 cycle); 60° C., 30 s, 72°    C., 3 min, 95° C., 30 s (30 cycles); 72° C., 10 min (1 cycle).

FIG. 20 depicts diagrammatically the cloned sequence section. Sequencingwas carried out in strand and counterstrand orientation, using thecloned fragments and the PCR products. FIG. 21 depicts the sequence ofthe cloned sequence section.

Sequence Comparisons

The nucleotide sequence of carB and the peptide sequence of the derivedprotein CarB were compared with the known sequences of related proteins.The sequences were compared using the GAP and BESTFIT programs.

CarB—Identical Aminoacyl Residues According to GAP

Program Settings: Gap weight: 8 Length weight: 2 Average match: 2.912Average mismatch: −2.003

The following values, in %, of amino acid correspondence to CarB ofBlakeslea trispora ATCC14272 were found: Phycomyces blakesleeanus:72.491 Phaffia rhodozyma: 50.460 Neurospora crassa: 47.943 Cercosporanicotianae: 47.740CarB—Identical Aminoacyl Residues According to BESTFIT

Program Settings: Gap weight: 8 Length weight: 2 Average match: 2.912Average mismatch: −2.003

The following values, in %, of amino acid correspondence to CarB ofBlakeslea trispora ATCC14272 were found: Phycomyces blakesleeanus:73.380 Phaffia rhodozyma: 53.175 Neurospora crassa: 51.896 Cercosporanicotianae: 50.791carB—Identical Bases According to GAP

Program Settings: Gap weight: 50 Length weight: 3 Average match: 10.000Average Mismatch: 0.000

The following values, in %, of base correspondence to CarB of Blakesleatrispora ATCC14272 were found: Phycomyces blakesleeanus: 64.853Cercospora nicotianae: 50.143 Phaffia rhodozyma: 43.179 Neurosporacrassa: 42.130carB—Identical Bases According to BESTFIT

Program Settings: Gap weight: 50 Length weight: 3 Average match: 10.000Average mismatch: −9.000

The following values, in %, of base correspondence to CarB of Blakesleatrispora ATCC14272 were found: Phycomyces blakesleeanus: 68.926 Phaffiarhodozyma: 62.403 Neurospora crassa: 60.230 Cercospora nicotianae:56.884Cloning for carB Expression

In order to clone and express Blakeslea trispora carB, the possibleprotein sequences were derived in six reading frames from theabove-described cloned sequence section from Blakeslea trispora. Theseprotein sequences were compared with the sequences of the phytoenedesaturates from Phycomyces blakesleeanus, Phaffia rhodozyma, Neurosporacrassa, Cercospora nicotianae. On the basis of the sequence comparison,three exons were identified in the cloned sequence section of theBlakeslea trispora genomic DNA, which, put together, result in a codingregion whose derived gene product has, over its entire length, 72.7%identical aminoacyl residues with the CarB phytoene desaturase ofPhycomyces blakesleeanus. This sequence section comprising threepossible exons and two possible introns was therefore referred to asgene carB. In order to check the predicted gene structure, the codingsequence of Blakeslea trispora carB was generated by means of PCR usingBlakeslea trispora cDNA as template and the primers Bol14255′-AGAGAGGGATCCTTAAATGCGAATATCGTTGC-3′ (SEQ ID 56) and Bol14265′-AGAGAGGGATCCATGTCTGATCAAAAGAAGCA-3′ (SEQ ID 57). The DNA fragmentobtained was sequenced. The location of exons and introns was confirmedby comparing the cDNA with the genomic carB DNA. FIG. 21 depictsdiagrammatically the coding sequence of carB. For expression of carB inEscherichia coli, first the NdeI cleavage site in carB was removed bythe overlap extension PCR method and an NdeI cleavage site wasintroduced at the 5′ end of the gene and a BamHI cleavage site wasintroduced at the 3′ end. The DNA fragment obtained was ligated with thevector pJOE2702. The plasmid obtained was referred to as pBT4 and clonedtogether with pCAR-AE into Escherichia coli XL1-Blue. Expression wasinduced with rhamnose. The enzyme activity was detected by way ofdetecting lycopine synthesis via HPLC. The cloning steps are describedbelow:

PCR 1.1:

Approx. 0.5 μg of Blakeslea trispora cDNA, 0.25 μM MAT3505′-ACTTTATTGGATCCTTAAATGCGAATATCGTTGCTGC-3′ (SEQ ID 58), 0.25 μM MAT2445′-GTTCCAATTGGCCACATGAAGAGTAAGACAGGAAACAG-3′ (SEQ ID 59), 100 μM dNTP,10 μl of Pfu polymerase buffer (10×), 2.5 U of Pfu polymerase (additionat 85° C., “hot start”) and H₂O ad 100 μL.

Temperature Profile:

1. 95° C. 10 min, 2. 85° C. 5 min, 3. 40° C. 30 s, 4. 72° C. 1 min 30 s,5. 95° C. 30 s, 6. 50° C. 30 s, 7. 72° C. 1 min 30 s, 8. 95° C. 30 s, 9.72° C. 10 min

Cycles: (1-2.) 1×, (3-5.) 5×, (6-8.) 25×, (9.) 1×

PCR 1.2:

Approx. 0.5 μg of Blakeslea trispora cDNA, 0.25 μM MAT2435′-CCTGTCTTACTCTTCATGTGGCCAATTGGAACCAACAC-3′ (SEQ ID 60), 0.25 μM MAT3535′-CTATTTTAATCATATGTCTGATCAAAAGAAGCATATTG-3′ (SEQ ID 61), 100 μM dNTP,10 μl of Pfu polymerase buffer (10×), 2.5 U of Pfu polymerase (additionat 85° C., “hot start”) and H₂O ad 100 μL.

Temperature Profile:

1. 95° C. 10 min, 2. 85° C. 5 min, 3. 40° C. 30 s, 4. 72° C. 1 min 30 s,5. 95° C. 30 s, 6. 50° C. 30 s, 7. 72° C. 1 min 30 s, 8. 95° C. 30 s, 9.72° C. 10 min

Cycles: (1-2.) 1×, (3-5.) 5×, (6-8.) 25×, (9.) 1×

Purification of the PCR Fragments from PCR 1.1, 1.2

For this purpose, PCR 2 was carried out to prepare the coding sequenceof Blakeslea trispora carB for cloning into pJOE2702:

Approx. 50 ng of PCR 1.1 product and approx. 50 ng of PCR 1.2 product,with 0.25 μM MAT350 5′-ACTTTATTGGATCCTTAAATGCGAATATCGTTGCTGC-3′ (SEQ IDNO 58), 0.25 μM MAT353 5′-CTATTTTAATCATATGTCTGATCAAAAGAAGCATATTG-3′ (SEQID NO 61), 100 μM dNTP, 10 μL of Pfu polymerase buffer (10×), 2.5 U ofPfu polymerase (addition at 85° C., “hot start”) and H₂O ad 100 μL.

Temperature Profile:

1. 95° C. 10 min, 2. 85° C. 5 min, 3. 59° C. 30 s, 4. 72° C. 2 min, 5.95° C. 30 s, 6. 72° C. 10 min

Cycles: (1-2.) 1×, (3-5.) 22×, (6.) 1×

Subsequently, the fragment obtained (˜1.7 kbp) was purified, followed byligation into the vector pPCR-Script-Amp, cloning into Escherichia coliXL1-Blue, sequencing of the insert, cleavage with NdeI and BamHI andligation into pJOE2702. The plasmid obtained was referred to as pBT4.

Characterization and Detection of the Enzyme Activity of CarB (PhytoeneDesaturase)

The gene product derived from carB was referred to as CarB. CarB has thefollowing properties, based on peptide sequence analysis: Length: 582aminoacyl residues Molecular mass: 66470 Isoelectric point: 6.7Catalytic activity: Phytoene desaturase Reactant: Phytoene Product:Lycopene EC number: EC 1.14.99-

The enzyme activity was detected in vivo. Transfer of the plasmid(pCAR-AE) into Escherichia coli XL1-Blue produces the strain Escherichiacoli XL1-Blue (pCAR-AE). This strain synthesizes phytoene. An additionaltransfer of the pBT4 plasmid into Escherichia coli XL1-Blue produces thestrain Escherichia coli XL1-Blue (pCAR-AE)(pBT4). Since an enzymiclyactive phytoene desaturase is formed starting from carB, this strainproduces lycopene.

The plasmids pCAR-AE and pBT4 were therefore transferred intoEscherichia coli. The carotenoids were extracted from the cells grown inliquid culture and characterized (cf. above).

HPLC analysis revealed that the Escherichia coli XL1-Blue (pCAR-AE)strain produces phytoene and the Escherichia coli XL1-Blue(pCAR-AE)(pBT4) strain produces lycopene. Consequently, CarB has theenzyme activity of a phytoene desaturase.

Preparation of Genetically Modified Blakeslea trispora Strains forProducing Phytoene

The preparation of genetically modified organisms for producing phytoeneis described by way of example below.

Vector pBinAHygΔcarB for Generating carB⁻ Mutants of Blakeslea trispora

The vector pBinAHygΔcarB (SEQ. ID. NO:62, FIG. 22) was constructed todelete carB in Blakeslea trispora. The precursor of pBinAHygΔcarB ispBinAHyg (SEQ. ID. NO:3, FIG. 2) which was constructed as follows:

The gpdA-hph cassette was isolated as BglII/HindIII fragment from theplasmid pANsCos1 (SEQ. ID. NO:4, FIG. 1, Osiewacz, 1994, Curr. Genet.26:87-90) and ligated into the BamHI/HindIII-opened binary plasmidpBin19 (Bevan, 1984, Nucleic Acids Res. 12:8711-8721). The vectorobtained in this way was referred to as pBinAHyg and comprises the E.coli hygromycin resistance gene (hph) under the control of the gpdpromoter and the trpc terminator from Aspergillus nidulans and theappropriate border sequences required for the Agrobacterium DNAtransfer.

The carB coding sequence was amplified by means of PCR using the primersMAT350 (SEQ ID NO 58) and MAT353 (SEQ ID NO 61) and the followingparameters: 50 ng of pBT4 with 0.25 μM MAT3505′-ACTTTATTGGATCCTTAAATGCGAATATCGTTGCTGC-3′, 0.25 μM MAT3535′-CTATTTTAATCATATGTCTGATCAAAAGAAGCATATTG-3′, 100 μM dNTP, 10 μl of Pfupolymerase buffer, 2.5 U of Pfu polymerase (addition at 85° C., “hotstart”) and H₂O to 100 μl

Temperature Profile:

1. 95° C. 10 min, 2. 85° C. 5 min, 3. 58° C. 30 s, 4. 72° C. 2 min, 5.95° C. 30 s, 6. 72° C. 10 min.

Cycles: (1.-2.) 1×, (3-5.) 30×, (6.) 1×

The fragment obtained (˜1.7 kbp) was subsequently purified, followed bycleavage with HindIII, further purification of the 364 bp HindIIIfragment carB, followed by cleavage of pBinAHyg with HindIII, ligationof the 364 bp HindIII fragment carB into pBinAHyg, transformation of thevector into Escherichia coli and isolation of the construct and referredto as pBinAHygΔcarB, as described above. Alternatively, partial cleavagewith HindIII was carried out and a larger carB HindIII fragment wascloned into pBinAHyg to produce pBinAHygΔcarB.

Generation of carB⁻ Mutants of Blakeslea trispora

The pBinAHygΔcarB plasmid was first transferred into the Agrobacteriumstrain LBA 4404, for example by electroporation (cf. above). The plasmidwas subsequently transferred from Agrobacterium tumefaciens LBA 4404 inBlakeslea trispora ATCC 14272 and in Blakeslea trispora ATCC 14271 (cf.above). Successful detection of the gene transfer into Blakesleatrispora was carried out via polymerase chain reaction according to thefollowing protocol:

approx. 0.5 μg of DNA from Blakeslea trispora ATCC 14272 carB⁻ or ATCC14271 carB⁻ was reacted with 0.25 μM primer hph forward5′-CGATGTAGGAGGGCGTGGATA-3′ (SEQ ID NO 5), 0.25 μM primer hph reverse5′-GCTTCTGCGGGCGATTTGTGT-3′ (SEQ ID NO 6), 100 μM dNTP, 10 μL ofHerculase polymerase buffer, 2.5 U of Herculase DNA polymerase (additionat 85° C., “hot start”) and H₂O to 100 μl.

Temperature Profile:

1. 95° C. 10 min, 2. 85° C. 5 min, 3. 58° C. 1 min, 4. 72° C. 1 min, 5.94° C. 1 min, 6. 72° C. 10 min.

Cycles: (1.-2.) 1×, (3-5.) 30×, (6.) 1×.

It was attempted to amplify the Agrobacterium kanamycin resistance geneas a negative control. For this purpose, the following PCR conditionswere used: approx. 0.5 μg of DNA from Blakeslea trispora ATCC 14272carB⁻ and ATCC 14271 carB⁻ was reacted with 0.25 μM primer nptIIIforward 5′-TGAGAATATCACCGGAATTG-3′ (SEQ ID NO 7), 0.25 μM primer nptIIIreverse 5′-AGCTCGACATACTGTTCTTCC-3′ (SEQ ID NO 8), 100 μM dNTP, 10 μL ofHerculase polymerase buffer, 2.5 U of Herculase DNA polymerase (additionat 85° C., “hot start”) and H₂O to 100 μl.

Temperature Profile:

1. 95° C. 10 min, 2. 85° C. 5 min, 3. 58° C. 1 min, 4. 72° C. 1 min, 5.94° C. 1 min, 6. 72° C. 10 min—

Cycles: (1-2.) 1×, (3-5.) 30×, (6.) 1×

C) Production of Carotenoids and Carotenoid Precursors by Blakesleatrispora

The carotenoids zeaxanthin, canthaxanthin, astaxanthin and phytoene wereproduced by fermenting the corresponding genetically modified Blakesleatrispora (+) and (−) strains, detecting the carotenoid produced by meansof HPLC analysis and isolating it.

The liquid medium for producing carotenoids comprised, per liter: 19 gof cornflour, 44 g of soybean flour, 0.55 g of KH₂PO₄, 0.002 g ofthiamine hydrochloride, 10% sunflower oil. The pH was adjusted to 7.5with KOH.

To produce the carotenoids, shaker flasks were inoculated with sporesuspensions of (+) and (−) strains of the Blakeslea trispora GMO. Theshaker flasks were incubated at 26° C. and 250 rpm for 7 days.Alternatively, trisporic acids were added to mixtures of the strainsafter 4 days, followed by 3 more days of incubation. The finalconcentration of the trisporic acids was 300-400 μg/ml.

Extraction and Analysis

Extraction:

-   1. Removal of 10 ml of culture suspension-   2. Centrifugation, 10 min, 5000×g-   3. Discarding of the supernatant-   4. Resuspension of the pellet in 1 ml of tetrahydrofuran (THF) by    vortexing-   5. Centrifugation, 5 min, 5000×g-   6. Removal of the THF phase-   7. Repetition of steps 4.-6. (2×)-   8. Pooling of the THF phases-   9. Centrifugation of the pooled THF phases at 20 000×g for 5 min in    order to remove residual aqueous phase.    Analysis

Phytoene Measurement by Means of HPLC Column: ZORBAX Eclipse XDB-C8, 5um, 150*4.6 mm Temperature: 40° C. Flow rate: 0.5 ml/min Injectionvolume: 10 μl Detection: UV 220 nm Stop time: 12 min Post run time: 0min Maximum pressure: 350 bar Eluent A: 50 mM NaH₂PO₄, pH 2.5 withperchloric acid Eluent B: Acetonitrile Gradient: Time [min] A [%] B [%]Flow [ml/min] 0 50 50 0.5 12 50 50 0.5

Extracts of the fermentation broth were used as matrix. Prior to HPLC,each sample was filtered through a 0.22 μm filter. The samples were keptcool and protected from light. In each case 50-1000 mg/l were weighedand dissolved in THF for calibration. The standard used was phytoenewhich has a retention time of 7.7 min under the given conditions.

Measurement of Lycopene, β-Carotene, Echinenone, Canthaxanthin,Cryptoxanthin, Zeaxanthin and Astaxanthin by Means of HPLC Column:Nucleosil 100-7 C18, 250*4.0 mm (Macherey & Nagel) Temperature: 25° C.Flow rate: 1.3 ml/min Injection volume: 10 μl Detection: 450 nm Stoptime: 15 min Post run time: 2 min Maximum pressure: 250 bar Eluent A:10% acetone, 90% H₂O Eluent B: Acetone Gradient: Time [min] A [%] B [%]Flow [ml/min] 0 30 70 1.3 10 5 95 1.3 12 5 95 1.3 13 30 70 1.3

Extracts of the fermentation broth were used as matrix. Prior to HPLC,each sample was filtered through a 0.22 μm filter. The samples were keptcool and protected from light. In each case 10 mg were weighed anddissolved in 100 ml of THF for calibration. The following carotenoidswith the following retention times were used as standard: β-carotene(12.5 min), lycopene (11.7 min), echinenone (10.9 min), cryptoxanthin(10.5 min), canthaxanthin (8.7 min), zeaxanthin (7.6 min) andastaxanthin (6.4 min) [see FIG. 23].

Production of Zeaxanthin by Genetically Modified Blakeslea trisporaStrains

Production of zeaxanthin by genetically modified organisms (GMO) ofBlakeslea trispora is described by way of example below.

The vector pBinAHygBTpTEF1-HPcrtZ was transferred into Blakesleatrispora by Agrobacterium-mediated transformation (see above). Ahygromycin-resistant clone was isolated and transferred to apotato-glucose agar plate (Merck KGaA, Darmstadt, Germany).

Starting from this plate, a spore suspension was prepared after threedays of incubation at 26° C. A 250 ml Erlenmeyer flask without bafflesand comprising 50 ml of growth medium (47 g/l cornflour, 23 g/l soybeanflour, 0.5 g/l KH₂PO₄, 2.0 mg/l thiamine-HCl, pH adjusted to 6.2-6.7with NaOH before sterilization) was inoculated with 1×10⁵ spores. Thispreculture was incubated at 26° C. and 250 rpm for 48 hours. For themain culture, a 250 ml Erlenmeyer flask without baffles and comprising40 ml of production medium was inoculated with 4 ml of the precultureand incubated at 26° C. and 150 rpm for 8 days. The production mediumcomprised 50 g/l glucose, 2 g/l caseine acid hydrolysate, 1 g/l yeastextract, 2 g/l L-asparagine, 1.5 g/l KH₂PO₄, 0.5 g/l MgSO₄×7H₂O, 5 mg/lthiamine-HCl, 10 g/l Span20, 1 g/l Tween 80, 20 g/l linoleic acid, 80g/l corn steep liquor. After 72 hours, kerosene was added at a finalconcentration of 40 g/l.

After harvesting the cultures, the remaining culture volume ofapproximately 35 ml was increased to 40 ml with water. Subsequently, thecells were disrupted in a high pressure homogenizer, type Micron Lab 40,APV Gaulin, 3× at 1500 bar.

The suspension comprising the disrupted cells was admixed with 35 ml ofTHF and incubated with shaking at 250 rpm and RT in the dark for 60 min.Then 2 g of NaCl were added and the mixture was incubated with shakingonce more. The extraction mixture was then centrifuged at 5000×g for 10min. The colored THF phase was removed and the cell mass was completelycolorless.

The THF phase was concentrated to 1 ml in a rotary evaporator at 30 mbarand 30° C. and then taken up again in 1 ml of THF. After centrifugationat 20 000×g for 5 min, an aliquot of the upper phase was removed andanalyzed by HPLC (FIG. 24, FIG. 23).

D) Work-Up and Isolation of the Carotenoids and the Foodstuff

The culture broths indicated above under A) were worked up as follows inorder to obtain highly pure carotenoids and a corresponding foodstuff.

The carotenoid content of the culture broths 1, 2, 3 was between 0.5 and1.5 g/l.

D1) Example According to Variant a) IIA and Variant b) IIA or IIB

The cultures having identical media (approx. 1 l in total) were combinedat the end of the cultivation period and homogenized with the aid of adisperser (Ultra.Turrax®).

The concentration of solids in the media 1 and 2 was 37 g/l and 11 g/l,respectively. The culture broth was dehydrated using a centrifuge. Ifthe cell concentrations and the solids content of the medium are high,the culture broth may also be processed further without priorsolid-liquid separation (medium 3: 127 g of solid/l). After previoushomogenization using a disperser (Ultra-Turrax®) and with constantstirring of the suspension, the cell mass was applied via a peristalticpump to the dryer. Injection into the cylinder of the laboratory spraydryer was carried out via a two-component nozzle having a diameter of2.0 mm, with 2 bar and 4.5 Nm³/h of nitrogen. The intake temperature wasapprox. 125° C. to 127° C. The drying gas was nitrogen at a flow rate of22 Nm³/h. The exhaust temperature was between 59° C. and 61° C. For eachof the three fermentation broths it was possible to precipitate flowableproduct on the cyclone of the spray dryer. The wall films in the chamber(where present) flaked off the vessel wall automatically and areclassified as unproblematic.

Between 8 and 100 g of powdery foodstuff were obtained which could beused directly as animal feedstuff. It comprised approx. 1-10%carotenoids based on dry weight. The residual moisture was less than 5%.

Example According to Variant b) IIC

D2) Extraction with Tetrahydrofuran

The cells of in each case 40 ml of culture broths 1, 2, 3 were disrupted3× at 1500 bar by a high pressure homogenizer, type Micron Lab 40, APVGaulin. In each case 20 ml of the suspensions comprising the disruptedcells were admixed with 20 ml of tetrahydrofuran and incubated withshaking at 30° C. in a rotary shaker at 200 rpm for 30 min. Then 2 g ofNaCl were added and the phases were separated by centrifuging at 5000×gfor 5 min. The THF phase was removed. Subsequently, the aqueous phasewas extracted once more with 20 ml of THF. The extracts were combined.The carotenoid concentration was quantified by HPLC.

D3) Extraction with Dichloromethane

The biomass was removed from the culture broth (200 ml) bycentrifugation at 5000×g in a laboratory centrifuge for 10 min.

The removed wet biomass (in each case approx. 10 g to 100 g) was admixedwith 10-100 ml of water in order to remove water-soluble components. Thebiomass was removed (laboratory centrifuge) and then sterilized withsteam (T=121, t=30 min, 1 bar) in an autoclave, whereby the cells weredisrupted.

25-250 g of dichloromethane were added to the cell debris and thecarotenoid was extracted from the biomass by shaking. The biomass wasremoved in a laboratory centrifuge.

A solvent exchange from dichloromethane to methanol was carried out, forwhich the carotenoid solution was kept at 40° C. to 60° C. for approx. 4hours and, over this period, admixed continuously with a total volume of20-200 ml of methanol. Dichloromethane was recovered as solvent in theprocess. First carotenoid crystals precipitated. Subsequently, thesolution was slowly cooled to approx. 10° C. over 6 h, with the size andnumber of carotenoid crystals increasing. The mother liquor was thenfiltered off and the carotenoid crystals were dried. Part of the motherliquor may be reused for solvent exchange. The other part is distilledand the methanol purified in this way is reused in the solvent exchange.

0.0.08 g to 0.24 g of carotenoid crystals were obtained whose purity(HPLC, cf. above) was 95%. The yield of carotenoid crystals was 80%based on the concentration of carotenoid in the biomass.

The removed, dichloromethane-wet biomass, after steam distillation, wasspray-dried (T_(I)=125° C., T_(E)=60° C.) and may be used as animal feedsupplement.

To this end, the cell mass, after previous homogenization using adisperser (Ultra-Turrax) and with constant stirring of the suspension,was applied via a peristaltic pump to the dryer.

Injection into the cylinder of the laboratory spray dryer was carriedout via a two-component nozzle having a diameter of 2.0 mm, with 2 barand 4.5 Nm³/h nitrogen. The intake temperature was approx. 125° C. to127° C. The drying gas was nitrogen at a flow rate of 22 Nm³/h. Theexhaust temperature was between 59° C. and 61° C. For each of the threefermentation broths it was possible to precipitate flowable product onthe cyclone of the spray dryer. The wall films in the chamber (wherepresent) flaked off the vessel wall automatically and were classified asunproblematic.

Approx. 2.5-25 g of powdery foodstuff were obtained which could be useddirectly as animal feedstuff. It comprised approx. 0.5%-1.5% carotenoidsbased on dry weight. The residual moisture was less than 5%.

The total yield of carotenoid (including the purified carotenoidfoodstuff) was approx. 95% based on the starting amount of carotenoid inthe culture broth.

1. A method for producing carotenoids or their precursors usinggenetically modified organisms of the Blakeslea genus, which methodcomprises the following steps: (i) transformation of at least one of thecells, (ii) homokaryotic conversion of the cells obtained in step (i) toproduce cells in which one or more genetic characteristics of the nucleiare all modified in an identical manner and said genetic modificationmanifests itself in the cells, and (iii) selection and reproduction ofthe genetically modified cell or cells, (iv) cultivation of thegenetically modified cells, (v) preparation of the carotenoid producedby the genetically modified cells or the carotenoid precursor producedby said genetically modified cells.
 2. The method according to claim 1,wherein the cells are from fungi of the Blakeslea trispora species. 3.The method according to claim 1, wherein a vector or free nucleic acidsare used in the transformation of step (i).
 4. The method according toclaim 3, wherein the vector employed in the transformation is integratedinto the genome of at least one of the cells.
 5. The method according toclaim 4, wherein the vector employed in the transformation comprises apromoter and/or a terminator.
 6. The method according to claim 3,wherein a vector comprising a gpd, pcarB, pcarRA and/or ptef1 promoterand/or a trpC terminator is employed in the transformation.
 7. Themethod according to claim 3, wherein a vector comprising a resistancegene is employed in the transformation.
 8. The method according to claim7, wherein the vector employed in the transformation comprises ahygromycin resistance gene (hph.
 9. The method according to claim 6,wherein the gpd promoter comprises the sequence SEQ ID NO:
 1. 10. Themethod according to claim 6, wherein the trpC terminator comprises thesequence SEQ ID NO:
 2. 11. The method according to claim 6, wherein theptef1 promoter comprises the sequence SEQ ID NO:
 35. 12. The methodaccording to claim 6, wherein the gpd promoter and the trpC terminatorare derived from Aspergillus nidulans.
 13. The method according to claim3, wherein the vector comprises the sequence SEQ ID NO:
 3. 14. Themethod according to claim 1, wherein the transformation is carried outusing agrobacteria, conjugation, chemicals, electroporation, bombardmentwith DNA-loaded particles, protoplasts or microinjection.
 15. The methodaccording to claim 1, wherein a mutagenic agent is employed in thehomokaryotic conversion of step (ii).
 16. The method according to claim15, wherein the mutagenic agent employed isN-methyl-N′-nitronitrosoguanidine (MNNG), UV radiation or X rays. 17.The method according to claim 1, wherein the selection is carried out bylabeling and/or selecting the mononuclear cells.
 18. The methodaccording to claim 1, wherein 5-carbon-5-deazariboflavin (darf) andhygromycin (hyg) or 5-fluororotate (FOA) and uracil and hygromycin areemployed in the selection.
 19. The method according to claim 3, whereinthe vector employed in the transformation includes genetic informationfor producing carotenoids or their precursors.
 20. The method accordingto claim 3, wherein the vector employed in the transformation includesgenetic information for producing carotenes or xanthophylls.
 21. Themethod according to claim 3, wherein the vector employed in thetransformation includes genetic information for producing astaxanthin,zeaxanthin, echinenone, β-cryptoxanthin, andonixanthin, adonirubin,canthaxanthin, 3-hydroxyechinenone, 3′-hydroxyechinenone, lycopene,β-carotene, α-carotene, lutein, phytofluene, bixin or phytoene.
 22. Amethod for providing at least one highly pure carotenoid and a foodstuffcomprising carotenoid-producing organisms and at least the onecarotenoid, which method comprises, after cultivation ofcarotenoid-producing genetically modified organisms of the Blakesleagenus according to claim 1, the following steps: I) removal of thebiomass, IA) optional washing of the biomass with a solvent in whichcarotenoids are not soluble, in particular water, IB) sterilization andcell disruption of the biomass, IC) optional drying and/or homogeneousdistribution, and II) partial extraction of the carotenoids from thedisrupted biomass by means of a carotenoid-dissolving solvent andseparation of said solvent from said biomass, IIA) 1) removal ofresidual solvent from the carotenoid-containing biomass, 2) optionalhomogeneous suspension of the biomass, with a biomass solid contentof >2% and <50%, 3) drying of the biomass or suspension for producingthe foodstuff, IIB) 1) crystallization of the carotenoids from thesolvent used and isolation of the carotenoid crystals, in particular byfiltration.
 23. The method according to claim 22, wherein the at leastone carotenoid is selected from the group consisting of carotenes andxanthophylls.
 24. The method according to claim 22, wherein the at leastone carotenoid is selected from the group consisting of astaxanthin,zeaxanthin, echinenone, β-cryptoxanthin, andonixanthin, adonirubin,canthaxanthin, 3-hydroxyechinenone, 3′-hydroxy-echinenone, lycopene,β-carotene, lutein, phytofluene, bixin and phytoene.
 25. The methodaccording to claim 22, wherein the at least one carotenoid isastaxanthin, zeaxanthin, bixin or phytoene.
 26. The method according toclaim 22, wherein sterilization and cell disruption are carried outusing steam or microwave radiation.
 27. The method according to claim22, wherein the carotenoids are extracted from the biomass usingdichloromethane or supercritical carbon dioxide or tetrahydrofuran. 28.The method according to claim 27, wherein the carotenoids dissolved insupercritical carbon dioxide are isolated directly or are taken up indichloromethane.
 29. The method according to claim 22, wherein thecarotenoids are extracted from the biomass in a one-stage or, ifappropriate, multistage process.
 30. The method according to claim 22,wherein solvents are removed from the biomass in step IIA1) using steamdistillation.
 31. The method according to claim 22, wherein drying instep IIA3) is carried out using spray drying or contact drying.
 32. Themethod according to claim 22, wherein crystallization in step IIB1) iscarried out by replacing the solvent gradually with a solvent in whichcarotenoids are not soluble.
 33. The method according to claim 32,wherein the solvent used is replaced with water or with a lower alcohol.34. The method according to claim 13, wherein the genetically modifiedorganism of the Blakeslea genus can be produced by transformation with avector which comprises a sequence selected from the group consisting ofSEQ ID NOs: 37-51 and SEQ ID NO:
 62. 35. A method for producing afoodstuff comprising organisms of the Blakeslea genus and at least onecarotenoid, which method comprises, after cultivation ofcarotenoid-producing genetically modified organisms of the Blakesleagenus according to claim 1, the following steps: I) homogeneoussuspension of the solids of the culture broth, and IIA) for a biomasssolid content of the culture broth of >2%: 1) optional concentration ofthe culture broth to give a solid content of <50%, and 2) drying of theculture broth to produce the foodstuff, or IIB) for a solid content of<2% of the culture broth, 1) concentration of the culture broth to givea solid content of >2% and <50%, and 2) drying of the suspension toproduce the foodstuff, or IIC) independently of the solid content of theculture broth, 1) removal of the biomass, 2) optional washing of thebiomass with solvents in which carotenoids are not soluble, inparticular water, 3) sterilization and cell disruption, 4) optionaldrying and homogeneous distribution, 5) partial extraction of thecarotenoids from the biomass using a carotenoid-dissolving solvent, 5a)removal of the carotenoid-containing biomass from thecarotenoid-containing solvent, 5b) removal of residual solvent from thebiomass, and 5c) drying of the biomass to produce the foodstuff, 6)crystallization of the carotenoids from the solvent used in 5a) andisolation of the carotenoid crystals, in particular by filtration. 36.The method according to claim 35, wherein the at least one carotenoid isselected from the group consisting of carotenes and xanthophylls. 37.The method according to claim 35, wherein the at least one carotenoid isselected from the group consisting of astaxanthin, zeaxanthin,echinenone, β-cryptoxanthin, andonixanthin, adonirubin, canthaxanthin,3-hydroxyechinenone, 3′-hydroxyechinenone, lycopene, β-carotene, lutein,bixin and phytoene.
 38. The method according to claim 35, wherein the atleast one carotenoid is astaxanthin, zeaxanthin, bixin or phytoene. 39.The method according to claim 35, wherein sterilization and celldisruption in step IIC3) are carried out using steam or microwaveradiation.
 40. The method according to claim 35, wherein the carotenoidsare extracted from the biomass in step IIC5) using dichloromethane orsupercritical carbon dioxide.
 41. The method according to claim 40,wherein the carotenoids dissolved in supercritical carbon dioxide areisolated directly or are taken up in dichloromethane.
 42. The methodaccording to claim 35, wherein the carotenoids are extracted from thebiomass in a one-stage or, if appropriate, multistage process.
 43. Themethod according to claim 35, wherein solvents are removed from thebiomass in step IIC5b) using steam distillation.
 44. The methodaccording to claim 35, wherein drying in any of steps IIA2), IIB2) andIIC5c) is carried out using spray drying or contact drying.
 45. Themethod according to claim 35, wherein crystallization in step IIC6) iscarried out by replacing the solvent gradually with a solvent in whichcarotenoids are not soluble.
 46. The method according to claim 45,wherein the solvent used is replaced with water or with a lower alcohol.47. The method according to claim 35, wherein the genetically modifiedorganism of the Blakeslea genus can be produced by transformation with avector which has a sequence selected from the group consisting of SEQ IDNOs: 37-51 and SEQ ID NO:
 62. 48. A foodstuff, in particular animalfeedstuff, which can be produced by the method of claim
 1. 49. A foodsupplement, in particular animal feed supplement, which can be producedby the method of claim
 1. 50. A method for producing the foodstuff andanimal feedstuff of claim 48 comprising a fermentation.
 51. A method forproducing the food supplement and animal feed supplement of claim 49comprising a fermentation.
 52. The method according to claim 35, whereinat least two products of the group consisting of foodstuff, foodsupplement, animal feedstuff and animal feed supplement can be obtainedfrom a fermentation.
 53. The method according to claim 35, wherein thecarotenoids are incorporated in the production of cosmetic,pharmaceutical, dermatological preparations, foodstuffs, foodsupplements, animal feedstuff or animal feed supplement.
 54. The methodaccording to claim 8, wherein the hygromycin resistance gene (hph) isfrom E. coli.
 55. The method according to claim 33, wherein the solventis replaced with methanol.
 56. The method according to claim 46, whereinthe solvent is replaced with methanol.